CN106701814B

CN106701814B - Method and application of regulating starch content in potato leaves

Info

Publication number: CN106701814B
Application number: CN201510469270.1A
Authority: CN
Inventors: 张鹏; 周文智; 何树涛
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2015-08-03
Filing date: 2015-08-03
Publication date: 2020-04-14
Anticipated expiration: 2035-08-03
Also published as: CN106701814A

Abstract

本发明涉及一种调节薯类叶片中淀粉含量的方法及应用。首次揭示通过改变SRD基因在薯类植物中的表达，可以显著调节薯类植物的淀粉性状，在植物品质的遗传改良上具有良好的应用前景。The invention relates to a method and application for regulating starch content in potato leaves. It was revealed for the first time that by changing the expression of SRD gene in tubers, the starch properties of tubers can be significantly regulated, and it has a good application prospect in the genetic improvement of plant quality.

Description

Method for adjusting starch content in potato leaves and application

Technical Field

The invention belongs to the field of biotechnology and botany, and particularly relates to a method for regulating starch content in potato leaves and application thereof.

Background

A tuber plant refers to a type of terrestrial crop having edible tuberous roots or subterranean stems. The method comprises the following steps of carrying out multi-row asexual propagation on tuberous roots and tubers, such as sweet potatoes (sweet potatoes and sweet potatoes), cassava, potatoes, yams (Chinese yams), and rhizoma podocarpi, wherein only the potato blocks are left for seeds and can be propagated by using lianas. The plants are generally weak in cold resistance, are usually cultivated in frostless seasons, and can inhibit the growth of the potato crops at low temperature to cause the yield reduction of root tubers or tubers, so that the long-time low-temperature period is avoided as much as possible when the potato crops are planted; in addition, loose, fertile and deep soil and a large amount of potash fertilizer are beneficial to improving the yield and the quality of potato crops.

Most of the stored roots and leaves of the potato plants are rich in starch in a large amount, the stored roots of the potato plants are the main raw materials for producing the starch at present, but the leaves are used as the main organs for fixing carbon dioxide by photosynthesis and also have the potential for producing novel starch. The production of novel starch by controlling the accumulation of starch in leaves and improving the properties of starch in leaves and fully exerting the advantages of potato leaves is always the focus of research in the field.

Disclosure of Invention

The invention aims to provide a method for adjusting the starch content in potato leaves and application thereof.

In a first aspect of the invention, there is provided a method of modulating starch traits in the leaves of a potato plant, the method comprising: modulating expression of an SRD polypeptide in a potato plant.

In a preferred embodiment, the potato plant comprises: cassava, sweet potato, yam, taro, kudzu root, konjak, jerusalem artichoke, yacon and the like.

In another preferred embodiment, the SRD polypeptide is selected from the group consisting of:

(a) 2 amino acid sequence of a polypeptide as set forth in SEQ ID NO;

(b) a polypeptide derived from (a) wherein the amino acid sequence of SEQ ID NO:2 is substituted, deleted or added with one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues, and which has the function of the polypeptide of (a); or

(c) A polypeptide derived from (a) having a homology of 70% or more (preferably 80% or more, 85% or more, more preferably 90% or more, such as 95% or more, 98% or more, or 99% or more) with the polypeptide sequence defined in (a) and having the function of the polypeptide of (a).

In another preferred example, the method comprises: reducing expression of an SRD polypeptide in a plant, thereby:

the starch content in the potato plant leaves is improved;

increasing the diameter of starch granules in the leaves of the potato plants;

reducing the phosphorylation degree of starch in the potato plant leaves;

increasing the amylose content in the leaves of the potato plants; and/or

The content of short chain (preferably DP6-14) in the starch is increased, and the content of long chain part (preferably DP20-37) in the starch is reduced.

In another preferred embodiment, said reducing expression of an SRD polypeptide in a plant comprises: transferring an interfering molecule that interferes with the expression of the SRD polypeptide into a plant (e.g., a cell, tissue, organ, or seed of a plant), thereby down-regulating the expression of the SRD polypeptide in the plant.

In another preferred embodiment, the interfering molecule that interferes with the expression of the SRD polypeptide targets the gene encoding the SRD polypeptide or a transcript thereof; preferably, the 741-1193 position of the coding gene or a transcript thereof is targeted.

In another preferred embodiment, the interfering molecule comprises a structure of formula (I):

Seq_{forward direction}-X-Seq_{Reverse direction}A compound of the formula (I),

in the formula (I), the compound is shown in the specification,

Seq_{forward direction}A fragment of a gene encoding an SRD polypeptide, Seq_{Reverse direction}Is and Seq_{Forward direction}A complementary polynucleotide; preferably, Seq_{Forward direction}741-1193 of a gene encoding an SRD polypeptide;

x is at Seq_{Forward direction}And Seq_{Reverse direction}A spacer sequence therebetween, and the spacer sequence and Seq_{Forward direction}And Seq_{Reverse direction}Are not complementary.

In another preferred embodiment, the structure of formula (I) forms a secondary structure of formula (II) after transfer into a plant:

in the formula (II), Seq_{Forward direction}、Seq_{Reverse direction}And X is as defined above,

i is expressed in Seq_{Forward direction}And Seq_{Reverse direction}Substantially complementary relationship therebetween.

In another preferred example, the method further comprises the subsequent steps of: selecting from the plants after modulating expression of the SRD polypeptide a plant that has acquired an altered trait as compared to the plant prior to modulation.

In another aspect of the invention, there is provided the use of an agent which down-regulates (e.g. interferes with) the expression of an SRD polypeptide for modulating starch traits in the leaves of a potato plant.

In a preferred embodiment, the agent which down-regulates expression of an SRD polypeptide is used to:

the starch content in the potato plant leaves is improved;

increasing the diameter of starch granules in the leaves of the potato plants;

reducing the phosphorylation degree of starch in the potato plant leaves;

increasing the content of amylose in the temporary starch of the potato plant leaves; and/or

The short chain content in the starch is increased, and the long chain part content in the starch is reduced.

In another preferred embodiment, the potato plant comprises: cassava, sweet potato, yam, taro, kudzu root, konjak, jerusalem artichoke, yacon and the like.

In another aspect of the invention, there is provided an interfering molecule which down-regulates the expression of an SRD polypeptide, thereby modulating the starch trait of the leaves of a potato plant, comprising the structure of formula (I):

Seq_{forward direction}-X-Seq_{Reverse direction}A compound of the formula (I),

in the formula (I), the compound is shown in the specification,

Seq_{forward direction}Is a gene segment, Seq, encoding an SRD polypeptide_{Reverse direction}Is and Seq_{Forward direction}A complementary polynucleotide; preferably, Seq_{Forward direction}741-1193 of a gene encoding an SRD polypeptide;

In another aspect of the invention, a vector is provided, said vector comprising said interfering molecule.

In another aspect of the invention, there is provided the use of an SRD polypeptide, or a gene encoding the same, as a molecular marker for identifying starch traits in leaves of a potato plant; the starch character of the leaf of the potato plant comprises:

starch content in potato plant leaves;

the diameter of starch granules in the potato plant leaves;

degree of phosphorylation of starch in potato plant leaves;

the amylose content in the temporary starch of the potato plant leaves; and/or

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a schematic diagram of an RNAi binary expression vector containing a hairpin structure.

FIG. 2, Southern blot identification of transgenic plants.

FIG. 3, change in expression level of SRD in SRDRNAi transgenic plants.

FIG. 4 Western blot analysis of MeSRD protein expression in transgenic plants.

Antibody: MeSRD rabbit polyclonal antibody;

internal reference: the Rubisco is taken as a protein loading internal reference;

comparison: wild-type cassava TMS 60444;

the protein extraction material is mature leaf of greenhouse cassava plant.

Figure 5, content of temporary starch in wild-type cassava TMS60444 and transgenic cassava leaves. The leaf is taken from the five top libraries of Shanghai to test cassava plants grown under the natural condition in the test field, and the data shown in the figure is 3 times of repeated experiments.

FIG. 6, dark cycle end wild type cassava TMS60444 and transgenic cassava leaf iodine staining results in different development periods.

FIG. 7, scanning electron microscope results of starch in wild type cassava TMS60444 and transgenic cassava leaves.

After the starch grains in the leaves are extracted, the starch grains are observed by a scanning electron microscope, and A-D are respectively starch in the leaves of WT, a transgenic strain G1i-12, a transgenic strain G1i-17 and a transgenic strain G1 i-31.

Fig. 8, phosphorylation level maps of starch in wild-type cassava TMS60444 and transgenic cassava leaves.

A: glucose-6-phosphate standard sample;

b: a glucose-3-phosphate standard sample;

c: the content of glucose-6-phosphate and glucose-3-phosphate in wild type cassava TMS60444 leaves;

d: the contents of glucose-6-phosphate and glucose-3-phosphate in SRDRNAi transgenic cassava leaves.

Fig. 9, phosphorylation degree of starch in wild-type cassava TMS60444 and transgenic cassava leaves.

The ordinate is the amount of glucose-6-phosphate contained per mg of starch, the starch extract material being obtained from the leaves of a Tagetes patula plant. The t-test-representative differences were very significant (p < 0.01).

Figure 10 amylose content of starch in wild-type cassava TMS60444 and transgenic cassava leaves.

Data shown in the figure are three replicates. Marked t-test differences (p <0.05) and marked t-test differences (p < 0.01).

Figure 11, XRD diffraction patterns of starch in wild-type tapioca TMS60444 and transgenic tapioca leaves.

Diffraction patterns of leaf transient starch; the starch is respectively extracted from the leaves and the root tuber of the cassava in the field.

FIG. 12, chain length distribution of starch in wild-type cassava TMS60444 and transgenic cassava leaves.

A: chain length distribution of wild type cassava TMS60444 leaf amylopectin;

B-F: respectively the length distribution difference of the wild-type TMS60444 tuberous root storage starch, the transgenic plants G1i-12, 17, 28 and 31 leaf temporary starch and the wild-type TMS60444 leaf temporary starch. The data above are triplicate experiments.

Detailed Description

Through intensive research, the inventor finds that the starch character of the potato plant can be obviously regulated by changing the expression of the SRD in the potato plant, and the SRD has a good application prospect in genetic improvement of plant quality.

As used herein, "potato plant" of the present invention, also referred to as "potato crop," refers primarily to a type of terrestrial crop having edible root tubers or subterranean stems. Including but not limited to: root tuber plants of Euphorbiaceae such as Manihot esculenta, root tuber plants of Convolvulaceae such as Ipomoea batatas, tuber plants of Solanaceae such as Solanum tuberosum, root tuber plants of Dioscoreaceae such as Dioscorea opposita, tuber plants of Araceae such as taro and Amorphophallus konjac, root tuber plants of Leguminosae such as Pueraria lobata, tuber plants of Compositae such as Jerusalem artichoke and yacon.

The invention also includes fragments, derivatives and analogs of the SRD polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an SRD polypeptide of the present invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more amino acid residues which are conserved or not (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide having an additional amino acid sequence fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

Any biologically active fragment of an SRD polypeptide can be used in the present invention. As used herein, a biologically active fragment of an SRD polypeptide is meant to be a polypeptide that retains all or part of the function of the full-length SRD polypeptide. Typically, the biologically active fragment retains at least 50% of the activity of the full-length SRD polypeptide. More preferably, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length SRD polypeptide.

In the present invention, the term "SRD polypeptide" refers to a polypeptide having the sequence of SEQ ID NO. 2 having the activity of an SRD polypeptide. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as the SRD polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of SRD polypeptides.

Variants of the polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to SRD polypeptide DNA under conditions of high or low stringency. The invention also provides other polypeptides, such as fusion proteins comprising an SRD polypeptide or fragment thereof.

Any protein having high homology to the SRD polypeptide (e.g., 70% or greater homology to the sequence shown in SEQ ID NO: 2; preferably 80% or greater homology; more preferably 90% or greater homology, e.g., 95%, 98% or 99% homology) and having the same function as the SRD polypeptide is also included in the present invention.

The induced variants may be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagenizing agent, site-directed mutagenesis, or other known molecular biological techniques, and analogs also include analogs having residues other than the natural L-amino acid (e.g., the D-amino acid), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).

It is to be understood that while the SRD polypeptides of the invention are preferably obtained from cassava, other polypeptides that are highly homologous (e.g., have greater than 70%, such as 80%, 90%, 95%, or even 98% sequence identity) to cassava SRD polypeptides obtained from other plants are also within the contemplation of the invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The present invention also relates to polynucleotide sequences encoding the SRD polypeptides of the invention or conservative variant polypeptides thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 1 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 2, but differ from the sequence of the coding region shown in SEQ ID NO. 1.

The polynucleotide encoding the mature polypeptide of SEQ ID NO. 2 comprises: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising a sequence encoding the polypeptide, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.

The full-length nucleotide sequence of the SRD gene of the invention or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates.

The invention also relates to vectors comprising said polynucleotides, and to genetically engineered host cells using said vectors or SRD gene sequences.

Based on the inventors' novel findings, the present invention also provides a method for improving a potato plant, comprising modulating expression of an SRD polypeptide in said potato plant.

More preferably, the method comprises: reducing expression of an SRD polypeptide in said plant (including causing no or low expression of an SRD polypeptide), thereby: the starch content in the potato plant leaves is improved; increasing the diameter of starch granules in the leaves of the potato plants; reducing the phosphorylation degree of starch in the potato plant leaves; and/or increasing the amylose content of the temporary starch of the potato plant leaves; the short chain content in the starch is increased, and the long chain part content in the starch is reduced.

Various methods known to those skilled in the art can be used to reduce or delete expression of an SRD polypeptide, such as delivering an expression unit (e.g., an expression vector or virus, etc.) carrying an antisense SRD gene to a target such that cells or plant tissues do not express or reduce expression of the SRD polypeptide.

As an embodiment of the present invention, there is provided a method of reducing expression of an SRD polypeptide in a plant, the method comprising:

(1) transferring the interfering molecules interfering with SRD gene expression into plant tissues, organs or seeds to obtain the plant tissues, organs or seeds transformed with the interfering molecules; and

(2) regenerating the plant tissue, organ or seed obtained in step (1) into which the interfering molecule has been transferred into a plant.

As a preferred example, the method comprises the steps of:

(i) providing an agrobacterium carrying a vector interfering with gene expression, said vector being selected from the group consisting of:

(a) a vector comprising a gene or gene fragment (antisense molecule) encoding a reverse-acting SRD polypeptide;

(b) a vector comprising an interfering molecule capable of forming a moiety that specifically interferes with the expression (or transcription) of a gene encoding an SRD polypeptide in a plant;

(ii) (ii) contacting a tissue or organ of the plant with the Agrobacterium of step (i) thereby transferring the vector into the plant tissue or organ.

Preferably, the method further comprises:

(iii) selecting a plant tissue or organ into which said vector has been transferred; and

(iv) (iv) regenerating the plant tissue or organ of step (iii) into a plant.

Based on the nucleotide sequence of the SRD gene, a polynucleotide can be designed which, when introduced into a plant, forms a molecule that specifically interferes with the expression of the SRD gene. The design takes into account specificity and efficiency of interference. The method for preparing the interfering molecule of the present invention is not particularly limited, and includes, but is not limited to: chemical synthesis, in vitro transcription, and the like. It is understood that, after knowing the association of the SRD gene with a plant trait, one skilled in the art can prepare the interfering molecules in various ways for use in modulating the plant trait. The interfering molecules can be delivered to the plant by transgenic techniques, or can also be delivered to the plant by a variety of techniques known in the art.

As a particularly preferred embodiment of the present invention, there is provided an interfering molecule having an excellent effect of specifically interfering with the expression of an SRD gene; and proved by verification, the gene has good effect of interfering SRD gene expression. The interfering molecule is a molecule containing a nucleotide sequence shown in the 741-1193 site in SEQ ID NO. 1, and forms a hairpin structure.

The invention also provides an interfering molecule comprising the following structure: seq_{Forward direction}Is an SRD gene segment (preferably the nucleotide sequence shown in the 741-1193 position in SEQ ID NO:1), SEQ_{Reverse direction}Is and Seq_{Forward direction}A complementary polynucleotide; x is at Seq_{Forward direction}And Seq_{Reverse direction}In betweenSpacer sequence, and said spacer sequence and Seq_{Forward direction}And Seq_{Reverse direction}Are not complementary.

The interfering molecule, when introduced into a plant, can fold into a stable stem-loop structure, the stem of the stem-loop structure comprising two substantially complementary sequences on either side of the stem. That is, a secondary structure is formed as follows:

wherein, | | is expressed in Seq_{Forward direction}And Seq_{Reverse direction}Substantially complementary relationship therebetween. The stem-loop structure can be further acted upon, processed or sheared by various substances in the plant body and forms double-stranded rna (dsrna).

Typically, the interfering molecule is located on an expression vector.

The present invention also includes plants obtainable by any of the methods described above, said plants comprising: transgenic plants into which an SRD gene or a homologous gene thereof has been transferred; or a plant having a reduced expression level (including low expression or no expression) of an SRD polypeptide, and the like.

The methods may be carried out using any suitable conventional means, including reagents, temperature, pressure conditions, and the like.

In addition, the invention also relates to the use of the SRD polypeptide or the gene encoding the SRD polypeptide as a tracking marker for progeny of genetically transformed plants. The invention also relates to the use of the SRD polypeptide or its coding gene as a molecular marker for identifying plant traits by detecting the expression of the SRD polypeptide in plants. The plant characteristics related to the SRD gene can also be used as an indicator mark of a true hybrid in the hybrid seed production process.

The method can prepare the potato plants with the improved starch content and the increased starch granules in the leaves, thereby realizing the process for extracting starch from the leaves of the potato plants in a large quantity.

In addition, the method can increase the diameter (3-10 μm) of starch granules in the leaves of the potato plants, so that the starch granules are closer to starch granules derived from beans (such as mung beans) or grains (such as rice), and the granular starch has good application value as small-granular starch (less than 10 μm).

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 cloning and sequence analysis of MeSRD

The inventor searches and finds two EST sequences in RIKEN cassava cDNA database (http:// www.brc.riken.jp/inf/en/index. shtml) through Blastp and Blastn, wherein the EST sequences are defined as MeSRD genes of cassava, the full length of the genes is 4230bp, and the EST sequences code for 1410 amino acids.

MeSRD nucleotide sequence (SEQ ID NO: 1):

ATGAGTAATAGCATAGGGCATAATTTATTCCAACAGAGTTTGATTCGTCCCGCGAGTTTTAAACATGGAAGCAATCTCAATTCTTCTGGCATTCCTGCAAGCTATTTATTCCAATCTGCCTCTGTGAGTCGAGGACTGCAGATAAGCAGGTCGCCAATATCCTCTAGTTTTTATGGAAAAAATTTGAGGGTGCGGAAATCAAAATTAGCCGTTGTAAATCCTCGTCCAGCTATAACAATTCCACGGGCTATATTGGCGATGGATCCGGCATCCCAGCTCCTAGGAAAATTCAACCTTGATGGAAATGTTGAATTGCAGGTGTTTGTTAGCAGTCACACTTCTGCTTCTACTGTGCAAGTACACATTCAGATAACATGCACTAGTGATTCTTTGCTCCTACACTGGGGTGGGAAACATGATAGAAAGGAAAACTGGGTACTTCCTAGTCGTTATCCAGATGGAACCAAAAATTATAAGAGCAGAGCCCTTAGAAGTCCTTTTGTCAAGTCTGGTTCAAGTTCTTACCTGAAAATAGAGATTGATGATCCTGCAATACAAGCCTTAGAATTTCTTATACTTGATGAAGCGCATAATAAATGGTTTAAAAATAATGGTGACAACTTTCATGTTAAATTACCTGCACGAGAGAAGCTGATAATTCCAAATATCTCAGTTCCTGAAGAGCTTGTACAAGTTCAAGCATATCTGAGGTGGGAAAGAAATGGTAAACAAATGTATACCCCAGAACAAGAAAAGAAAGAA TATGAAGCCGCTCGTATTGAACTATTGGAGGAAGTAGCTAAGGGTACTTCCATTGAGGGCCTCCGAGCAAGGCTGAC AAACAAAAATGAAATTAAGGAGTCATCTGTCTCTAAAACACAAAGCAAGATACACGCTCAAGCTCATAGAAGATGGG AAAAATCTACTACTAGTAATGAAAGGTTTCAGCGCAATCAGAGGGACTTAGCACAGCTTGTTACCAAATCTGCTACT AAAAAATCTGCAGAGGAAGCTGTTTCAGTAGAACCAAAACCAAAAGCATTGAAGGCAGTTGAACTTTTTGCTAAAGA AAAGGAAGAACGGGTTGGGGGTGCTGTTCTGAACAAGAAGATCTTTAAGCTCCAAGATGCGGAACTTCTGGTGCTTG TGACCAAGCCTGCTGATAAGATGAAGGTTTATGTTGCCACTGATTTCAAAGAACCAGTCACTCTTCACTGGGCATTATCTAGGAAGGGTAAAGAGTGGTTGGCGCCACCACCAAGTGTGTTGCCTCCTGGTTCAGTTTCTTTGAACGAGGCTGCTGAAACACAACTTAAAAGCATTTCTTCAACTGAACTTTCTTATCAGGTCCAATACTTTGAAACGGAGATCGAAGAGAATTTTGTAGGGATGCCCTTTGTGCTTTTTTCTAATGAAAAATGGATAAAGAATAAGGGCTCTGACTTTTATGTTGAACTTAGTGGCGGACCTAGGCCAGTCCAAAAGGATGCTGGTGATGGAAGAGGTACAGCAAAAGTTTTATTGGACACAATTGCAGAGCTGGAGAGTGAAGCACAGAAATCCTTCATGCACCGATTTAATATTGCAGCTGATTTGATGGAGGATGCAAAGGATGCTGGTGAGTTGGGTTTTGCAGGGATCTTGGTGTGGATGAGATTTATGGCCACGAGGCAACTTATTTGGAACAAAAACTACAATGTGAAACCACGTGAGATCAGCAAGGCACAGGATAGGCTCACAGACTTGCTCCAGAATACTTATACAAGTCATCCTCAATATCGGGAGCTTTTGCGGATGATTATGTCTACTGTCGGTCGAGGTGGTGAAGGTGATGTGGGGCAGCGAATTCGGGATGAAATTTTAGTTATCCAGAGAAACAATGATTGCAAAGGTGGTATGATGGAGGAATGGCATCAGAAGCTGCATAATAACACAAGCCCTGATGATGTTGTTATCTGCCAGGCATTAATGGATTACATTAAAAGTGACCTTGACATCAGTGTGTACTGGAAAACTTTGAATGAAAATGGAATAACAAAAGAACGACTTTTAAGCTATGATCGTGCAATCCATTCTGAACCAAGCTTCAGGAGAGATCAAAAGGACGGTCTTTTGCGTGATCTCGGCAACTATATGAGAAGTTTGAAGGCAGTTCATTCTGGTGCAGATCTTGAGTCTGCTATTGCAAATTGTATGGGCTATAAAGATGAGGGTCAAGGTTTCATGGTTGGAGTGCAAATAAATCCCATTTCAGGCTTGCCATCTGGATTTCCAGAGTTGCTTCGATTTGTTCTCAAACATGTTGAAGATAGAAATGTAGAAGCACTTCTTGAGGGTTTGCTGGAGGCTCGTCAGGAGCTGAGGCCATTGCTGTTTAAGTCTAATAATCGTCTGAAAGATCTTCTATTTTTGGATATTGCCCTTGATTCTACTGTTAGGACAGCCATTGAGAGAGGATATGAGGAATTAAATGATGCTGGACCAGAGAAAATTATGTATTTCATCACCCTGGTTCTTGAAAATCTTGCGCTTTCATCAGATGATAATGAAGAGTTTGTCTATTGCTTGAAGGGATGGAATTATGCCCTAAGCATGTCCAAAAGTAAAAGCAATCACTGGGCATTATATGCAAAATCAGTCCTTGACAGAACTCGCCTTGCCCTGGCCAGCAAGGCTGAATGGTATCAGCAAGTTTTGCAACCATCAGCAGAGTATCTTGGATCACTGCTTGGAGTGGATCAGTGGGCTGTGAACATATTCACTGAAGAAATAGTTCGTGCTGGATCAGCTGCAGCTGTATCCTTGCTTCTTAATCGACTTGATCCAGTTCTTCGGAAGACTGCTCATCTTGGAAGTTGGCAGGTTATTAGCCCAGTTGAAGCTGCTGGGTATGTTGTTGTTGTGGATGAGTTGCTCACAGTACAGAATTTATCTTACGACCGCCCTACAATTTTAGTGGCAAGAAGAGTAAGTGGAGAAGAAGAAATTCCTGATGGTACAGTTGCTGTGCTGACATCTGACATGCCAGATGTCCTATCCCATGTTTCTGTACGAGCAAGAAATAGCAAGGTTTGCTTTGCCACATGTTTTGATCACAACATTCTGGACAATCTCCGAGCAAATGAAGGGAAATTATTGAATTTGAAACCTACATCAGCAGATATAGTCTATAGCGTGATCGAGGGTGAATTAGCAGATTTAAGTTCAAATAAGCTGAAAGAAGTTGGTCCTTCACCTATAAAGTTGATAAGAAAGCAGTTCAGTGGTAGATATGCCATATCATCGGAGGAGTTCACCGGTGAAATGGTTGGTGCCAAATCACGCAATATCGCGCATCTAAAAGGAAAAGTACCATCCTGGATTGGGATTCCTACATCGGTTGCCTTACCATTTGGAGTTTTTGAGAAGGTTCTTTCAGATGGTTCAAATCAAGAAGTGGCTAAGAAGTTGGAAGTTTTGAAGAAACAGTTGGAAGGAGGAGAGTCTAGTGTCCTCAGGAGAATTCGTGAGACAGTTTTACAGCTGGCAGCACCACCACAGCTGGTGCAAGAGCTGAAGACAAAGATGAAAAGTTCTGGGATGCCTTGGCCTGGCGATGAAGGTGAACAGCGATGGGAGCAAGCATGGATGGCTATAAAGAAGGTCTGGGCTTCAAAATGGAATGAGAGAGCATACTTCAGCACAAGGAAAGTGAAGTTGGACCATGATTACCTCTGCATGGCTGTCCTGGTTCAGGAGATAATAAATGCCGATTATGCATTTGTTATCCACACGACCAATCCATCTTCTGGGGATTCATCAGAGATATATGCTGAGGTAGTGAAGGGACTTGGAGAAACTCTTGTTGGAGCCTATCCCGGCCGTGCTTTGAGTTTTATCTGCAAGAAAAAAGATCTGAATTCTCCTCAGGTGTTGGGTTACCCAAGCAAACCCATTGGCCTTTTTATAAGACGTTCTATAATCTTCAGATCTGACTCCAATGGTGAAGATCTGGAAGGTTATGCTGGTGCTGGTCTTTATGATAGTGTTCCAATGGATGAGGAAGAGAAAGTTGTGCTTGATTACTCATATGATCCATTGATCACCGATGAAAGCTTCCGAAAATCAATTCTCTCTAACATAGCTCGTGCTGGAAGTGCCATTGAAGAGCTCTATGGATCTCCACAAGACATTGAAGGAGTAATAAGGGACGGTAAACTCTATGTGGTTCAGACAAGGCCTCAGATGTAA

MeSRD amino acid sequence (SEQ ID NO: 2):

MSNSIGHNLFQQSLIRPASFKHGSNLNSSGIPASYLFQSASVSRGLQISRSPISSSFYGKNLRVRKSKLAVVNPRPAITIPRAILAMDPASQLLGKFNLDGNVELQVFVSSHTSASTVQVHIQITCTSDSLLLHWGGKHDRKENWVLPSRYPDGTKNYKSRALRSPFVKSGSSSYLKIEIDDPAIQALEFLILDEAHNKWFKNNGDNFHVKLPAREKLIIPNISVPEELVQVQAYLRWERNGKQMYTPEQEKKEYEAARIELLEEVAKGTSIEGLRARLTNKNEIKESSVSKTQSKIHAQAHRRWEKSTTSNERFQRNQRDLAQLVTKSATKKSAEEAVSVEPKPKALKAVELFAKEKEERVGGAVLNKKIFKLQDAELLVLVTKPADKMKVYVATDFKEPVTLHWALSRKGKEWLAPPPSVLPPGSVSLNEAAETQLKSISSTELSYQVQYFETEIEENFVGMPFVLFSNEKWIKNKGSDFYVELSGGPRPVQKDAGDGRGTAKVLLDTIAELESEAQKSFMHRFNIAADLMEDAKDAGELGFAGILVWMRFMATRQLIWNKNYNVKPREISKAQDRLTDLLQNTYTSHPQYRELLRMIMSTVGRGGEGDVGQRIRDEILVIQRNNDCKGGMMEEWHQKLHNNTSPDDVVICQALMDYIKSDLDISVYWKTLNENGITKERLLSYDRAIHSEPSFRRDQKDGLLRDLGNYMRSLKAVHSGADLESAIANCMGYKDEGQGFMVGVQINPISGLPSGFPELLRFVLKHVEDRNVEALLEGLLEARQELRPLLFKSNNRLKDLLFLDIALDSTVRTAIERGYEELNDAGPEKIMYFITLVLENLALSSDDNEEFVYCLKGWNYALSMSKSKSNHWALYAKSVLDRTRLALASKAEWYQQVLQPSAEYLGSLLGVDQWAVNIFTEEIVRAGSAAAVSLLLNRLDPVLRKTAHLGSWQVISPVEAAGYVVVVDELLTVQNLSYDRPTILVARRVSGEEEIPDGTVAVLTSDMPDVLSHVSVRARNSKVCFATCFDHNILDNLRANEGKLLNLKPTSADIVYSVIEGELADLSSNKLKEVGPSPIKLIRKQFSGRYAISSEEFTGEMVGAKSRNIAHLKGKVPSWIGIPTSVALPFGVFEKVLSDGSNQEVAKKLEVLKKQLEGGESSVLRRIRETVLQLAAPPQLVQELKTKMKSSGMPWPGDEGEQRWEQAWMAIKKVWASKWNERAYFSTRKVKLDHDYLCMAVLVQEIINADYAFVIHTTNPSSGDSSEIYAEVVKGLGETLVGAYPGRALSFICKKKDLNSPQVLGYPSKPIGLFIRRSIIFRSDSNGEDLEGYAGAGLYDSVPMDEEEKVVLDYSYDPLITDESFRKSILSNIARAGSAIEELYGSPQDIEGVIRDGKLYVVQTRPQM*

example 2 construction of MeSRDRNAi vector and obtaining of transgenic cassava

The present inventors first constructed an RNA interference (RNAi) binary vector specifically inhibiting MeSRD expression by selecting a MeSRD (pdm02348) gene specific fragment (741-1193 bp): pPCaMV35S: SRDRNAi.

Cassava (manihot utilissima) genome is taken as a template, and 5' -AT is takenGGTACCCCCAGAACAAGAAAAGAAAGAA-3 '(SEQ ID NO:3) and 5' -CTATCGATAAATCAGTGGCAACATAAACCT-3' (SEQ ID NO:4) to obtain a gene specific fragment (741-1193bp) of MeSRD (pdm02348), and inserting the forward fragment of the amplified MeSRD (741-1193bp) into the KpnI/ClaI cleavage site of pRNAi-dsAC1 binary vector (see Biotechnology and Bioengineering, Vol.108, No.8, August,2011, 1925-1935); with 5' -ATGGATCCCCCAGAACAAGAAAAGAAAGAA-3 '(SEQ ID NO:5) and 5' -CTCTCGAGAAATCAGTGGCAACATAAACCT-3' (SEQ ID NO:6) as a primer, a gene specific fragment (741-1193bp) of MeSRD (pdm02348) is obtained by amplification, and the amplified reverse repeated fragment of MeSRD (741-1193bp) is inserted into the XhoI/BamHI enzyme cutting site of the pRNAi-dsAC1 binary vector into which the forward fragment is inserted, so as to obtain the RNAi recombinant vector containing the hairpin structure.

The constructed RNAi recombinant vector is transferred into agrobacterium LBA4404, cassava brittle suspension callus is infected through agrobacterium, positive plants are obtained through regeneration, screening and other processes of the infected callus, and pPCaMV35S, namely SRDRNAi transgenic cassava is marked as SRDRNAi, which is abbreviated as G1 i.

Example 3 molecular characterization of MeSRDRNAi transgenic cassava

20 strains of SRDRNAi positive transgenic cassava G1i-1, 5, 12, 16, 17 and the like are obtained in total through agrobacterium-mediated cassava suspension callus transformation, and then single-copy transgenic plants are finally obtained through Southern blot screening, namely eleven strains of G1i-12, 17, 18, 22, 23 and the like are obtained in total.

(1) Identification of Gene expression levels

To verify the effect of RNA interference, the present inventors analyzed the expression level of MeSRD in single copy plants by Real-time RT-PCR using 5'-ACCTCTGCATGGCTGTCCTGGTT-3' (SEQ ID NO:7) and 5'-GCACGGCCGGGATAGGCTCC-3' (SEQ ID NO:8) as primers.

The results show that in most of pPCaMV35S, the expression level of MeSRD is obviously reduced in SRDRNAi single-copy transgenic cassava, wherein the reduction is most obvious in G1i-2, 12, 18, 28 and 31, and the expression level of MeSRD is reduced by 90% in the G1i-12 transgenic line compared with the wild-type cassava TMS60444, as shown in FIG. 3.

(2) Protein level expression identification

Although the target gene is strongly inhibited at the expression level, it is further proved whether the target gene is also inhibited at the protein level. The present inventors designed antigenic polypeptides against protein fragments specific for MeSRD and succeeded in obtaining rabbit-derived polyclonal antibodies to cassava MeSRD. Three leaves with the same size of wild cassava and MeSRD gene expression obviously reduced (G1i-12, G1i-17 and G1i-28) are harvested, and after total protein is extracted, the protein amount of MeSRD is further detected by Westernblot. Rubisco is taken as a protein loading internal reference, and wild cassava TMS60444 is taken as a control (WT); the protein extraction material is mature leaf of greenhouse cassava plant.

The results show that there is a target band of MeSRD in the wild type, with a size of about 140kD, consistent with the predicted MeSRD size; the transgenic plants G1i-12, 17 and 18 do not have target bands of MeSRD, which shows that the inventor successfully reduces the protein amount of the target gene by means of RNAi, and the expression level of the target gene is reduced, as shown in FIG. 4.

The above results indicate that the MeSRDRNAi transgenic cassava with the MeSRD expression effectively interfered is successfully obtained.

Example 4 Regulation of MeSRD on the temporary starch content of leaves

Respectively culturing wild cassava TMS60444 and SRDRNAi transgenic cassava in the Shanghai five-database pilot plant field under natural conditions. Mature leaves of 6:00, 12:00, 18:00 and 0:00 in the photoperiod are respectively taken, soluble polysaccharide is removed by methanol, and the Starch content in the leaves is measured by a Total Starch (K-TSTA, Megazyme) kit after drying. As a result, as shown in FIG. 5, the starch content of the wild-type cassava leaves gradually increased at the beginning of the photoperiod, the starch rapidly accumulated between 6:00 and 12:00, and the starch accumulation rate became relatively slow between 12:00 and 18:00, and the starch content reached the maximum of about 2% by the end of the photoperiod; starch degradation began as the dark cycle began, starch content gradually decreased, the rate of starch degradation remained essentially constant throughout the dark cycle, and starch was essentially completely reduced by the end of the dark cycle (6: 00). In the leaves of SRDRNAi transgenic cassava, the starch content is kept at a high level in the photoperiod, which is about 11 percent, and the fluctuation of the starch content in the photoperiod is not regular.

After the dark period is finished, wild-type cassava leaves and transgenic cassava leaves in different development periods are taken, alcohol is used for decoloring, and then the wild-type cassava leaves and the transgenic cassava leaves are dyed by 0.2% iodine solution, so that the wild-type cassava leaves cannot be colored, the transgenic cassava leaves in different periods are obviously blue, and the wild-type cassava leaves do not accumulate starch any more at the end of the dark period, but the transgenic cassava leaves still contain a large amount of starch, as shown in figure 6.

Example 5 Effect of MeSRD on leaf temporary starch morphology

Respectively culturing wild cassava TMS60444 and SRDRNAi transgenic cassava in the Shanghai five-database pilot plant field under natural conditions until the harvest period. Starch in wild-type cassava TMS60444 and SRDRNAi transgenic cassava leaves is respectively extracted, and the forms of the starch in the leaves and root tubers are observed through a Scanning Electron Microscope (SEM), so that the starch in the leaves is in a round cake shape, the diameter of the starch in the wild-type cassava leaves is about 2-3 mu m, starch granules in SRDRNAi transgenic plants are generally enlarged, the diameter of most of the starch can reach more than 5 mu m, and the diameter of the starch in transgenic plants G1i-12 and 17 with partial MeSRD expression down-regulated can reach about 10 mu m at most, as shown in figure 7.

The enlargement of starch grains not only can reduce the cost of the starch extraction process, but also is closer to the small starch grains of mung bean and grain sources.

Example 6 Regulation of the degree of transient starch phosphorylation in leaves by MeSRD

Respectively culturing wild cassava TMS60444 and SRDRNAi transgenic cassava in the Shanghai five-database pilot plant field under natural conditions until the harvest period. Collecting the light period terminal mature cassava leaves, extracting starch in the leaves, and analyzing the content of glucose-6-phosphate in hydrolysate through HPAEC-PAD after acid hydrolysis.

The chromatogram shows that the G-6-P and G-3-P standard products respectively show elution peaks in 22.7min and 24.2min, and obvious elution peaks of G-6-P and G-3-P are also detected in the temporary starch hydrolysate of the leaf blade of the wild cassava. Similar to other species, the content of G-3-P is obviously less than that of G-6-P, while the elution peak of G-6-P in temporary starch of leaves of RNAi transgenic plants is very weak, about 0.5% of that of wild cassava, which indicates that MeSRD in cassava also has the function of catalyzing phosphorylation of glucose residue C-6 in starch, as shown in FIG. 8. The degree of phosphorylation of starch in G1i-12, 17, 18 was most significantly down-regulated in MeSRDRNAi transgenic cassava plants, about 1ng/mg starch, and in G1i-28, 31, respectively, 7ng/mg starch and 15ng/mg starch, whereas the level of starch phosphorylation in wild-type leaves was 20ng/mg starch, which was essentially positively correlated to the expression level of MeSRD gene, as shown in FIG. 9.

Example 7 Regulation of amylose content in leaf temporary starch by MeSRD

As the starch form in the leaves changes, the amylose content also changes significantly.

And respectively culturing the wild type and the SRDRNAi transgenic cassava under the natural conditions of the Shanghai five-base pilot plant field, and culturing to the harvest stage. The amylose content of the leaf transient starch is determined.

The determination method comprises the following steps: the determination of the amylose content in the cassava starch is carried out according to national standard GB/T15683-2008/ISO 6647-1 of the people's republic of China: 2007. the method mainly comprises the following steps: weighing 50mg +/-0.5 mg of cassava starch, an amylose standard sample (Sigma, St.Louis, MO, USA) and an amylopectin standard sample (Sigma, St.Louis, MO, USA) in a test tube, adding 500 mu L of 95% ethanol to wash down a sample adhered to the inner wall of the test tube, shaking up gently, adding 4.5mL of 1.0M sodium hydroxide solution, mixing uniformly, carrying out boiling water bath for more than 10min until amyloid is completely dispersed, cooling to room temperature, and transferring to a 50mL volumetric flask for constant volume; preparing a blank without adding any amyloid according to the same step; preparing 1mL of series standard content solutions with amylose content of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% by using the standard amylose and amylopectin samples with constant volume for later use; diluting 8mL of 2% iodine solution, adding 4mL of 1.0M HAC solution, and diluting to a constant volume of 200mL to obtain a starch color developing solution, wherein the starch color developing solution is used as it is, and the same color developing solution is used for the same batch of samples; adding 100 μ L of blank control, tapioca amyloid and standard content solution into 1mL of starch color development solution, mixing uniformly for color development, taking 4 times of blank control and 3 times of other samples, and measuring OD value of 720nm wavelength on a spectrophotometer Libra S22(Biochrom Ltd., UK) after color development; and performing unary linear regression by using the absorbance value of the standard content solution at 720nm, solving a correlation coefficient, establishing an equation of the amylose content and the absorbance, and substituting the absorbance of the cassava amyloid to be detected into the equation to solve the amylose content of the cassava amyloid.

As a result, as shown in FIG. 10, the amylose content in the leaves of the transgenic plants is increased very significantly compared with the temporary starch of the wild-type cassava leaves, the amylose content in the wild-type cassava leaves is about 9%, and the amylose content in the temporary starch of the leaves of the SRDRNAi transgenic plants is increased to 22-37%, which is similar to the amylose content of the root tuber storage starch.

Example 8 adjustment of the thermodynamic Properties of leaf temporary starch by MeSRD

Differential Scanning Calorimetry (DSC) is a thermodynamic method for determining the endothermic capacity of starch during heating, which is mainly influenced by the amylose content, the crystallinity of starch, the amylopectin chain length distribution, etc. And (3) culturing wild type and SRDRNAi transgenic cassava under the natural condition of the Shanghai five-library pilot plant field, culturing to the harvest period, and performing thermodynamic property determination.

The thermodynamic property determination method comprises the following steps: a Q2000 differential scanning calorimeter (TA Instruments, Norwalk, CT, USA) was used for the thermodynamic analysis of starch sample gelatinization. The sample (10mg starch plus 30 μ L water, empty pan as blank control) was sealed in an alumina crucible and allowed to equilibrate at room temperature for 24h, then warmed from 30 ℃ to 95 ℃ at a rate of 10 ℃/min and the heat was scanned for changes. Data were analyzed using the Universal Analysis software.

The results are shown in table 1, in the transgenic plant of SRDRNAi, the leaf-temporal starch To (initial gelatinization temperature), Tp (peak gelatinization temperature) and the wild-type leaf-temporal starch are not obviously different, but Tc (representing end gelatinization temperature) and Δ H (gelatinization enthalpy) are obviously improved, wherein Δ H is about four times higher than that of the wild-type.

TABLE 1 thermodynamic parameters of starch in wild-type and transgenic cassava leaves

Note: the data shown in the figure are the mean ± sd of 3 experimental replicates. Values in each set of data labeled with the same letter (a, b, c, d, e) indicate that the measurements were not significantly different under these conditions (p < 0.05).

Example 9 Regulation of leaf temporary starch Structure by MeSRD

Culturing wild type and SRDRNAi transgenic cassava under the natural condition of the Shanghai five-library pilot test field, culturing to the harvest period, collecting cassava leaves, extracting temporary starch from the leaves, and measuring the diffraction pattern of the temporary starch of the leaves by using an X-ray diffractometer.

The XRD diffraction pattern of starch can be classified into A, B, C, V type starch, and is generally considered to be composed of diffraction peaks of crystalline regions and background peaks of amorphous regions. A starch XRD pattern in a wild type cassava leaf shows that the diffraction pattern has a plurality of characteristic peaks of crystalline regions, but background peaks of non-crystalline regions are weaker, which indicates that temporary starch non-crystalline regions in the wild type cassava leaf are fewer and the crystallinity is higher; the temporary starch in the SRDRNAi transgenic plant leaf has the same composition of a crystalline region and an amorphous region as common starch, the diffraction peaks of the temporary starch are mainly expressed as 15 degrees, 17 degrees and 23 degrees, the temporary starch is standard C-type starch and is similar to the diffraction pattern of storage starch, and the diffraction pattern is shown in figure 11.

In addition, the leaf transient starch was hydrolyzed by Isoamylase (Isoamylase), and then the glucan component in the hydrolysate was analyzed by HPAEC-PAD.

As shown in FIG. 12, the analysis result showed that the shortest glucan chain in the temporary starch of wild-type cassava leaves was DP6, two peaks at DP11-12 and DP45, and one shoulder at DP 18-20. Unlike the chain length distribution of tapioca leaf starch, the short chain content in root tuber storage starch is increased (2.2%) and the medium chain content is decreased (-0.8%). Compared with the wild type, the transgenic plant leaf starch has the chain length distribution change trend similar to the root tuber starch change: the short chain (DP6-14) is obviously increased by 0.4% at most; the mid-chain fraction (DP20-37) was significantly reduced, at about-0.2%.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method for increasing the diameter of starch granules in the leaves of a potato plant, increasing the content of short chains in starch and decreasing the content of long chain parts in starch, comprising: reducing expression of an SRD polypeptide in a potato plant.

2. The method of claim 1, wherein the potato plant comprises: cassava, sweet potato, yam, taro, kudzu root, konjak, jerusalem artichoke and yacon.

3. The method of claim 1, wherein said SRD polypeptide is a polypeptide having the amino acid sequence set forth in SEQ ID No. 2.

4. The method of claim 3, wherein said reducing expression of an SRD polypeptide in a plant comprises: transferring an interfering molecule that interferes with the expression of the SRD polypeptide into the plant, thereby down-regulating the expression of the SRD polypeptide in the plant.

5. The method of claim 4, wherein the interfering molecule that interferes with the expression of the SRD polypeptide targets position 741-1193 or a transcript thereof of a gene encoding the SRD polypeptide.

6. The method of claim 4, wherein the interfering molecule comprises a structure of formula (I):

Seq_{forward direction}-X-Seq_{Reverse direction}A compound of the formula (I),

in the formula (I), the compound is shown in the specification,

Seq_{forward direction}A fragment of a gene encoding an SRD polypeptide, Seq_{Reverse direction}Is and Seq_{Forward direction}A complementary polynucleotide;

7. The method of claim 6, wherein Seq_{Forward direction}Is the 741-1193 position of the gene encoding the SRD polypeptide.

8. The method of claim 1, further comprising the subsequent steps of: selecting from the plants after modulating expression of the SRD polypeptide a plant that has acquired an altered trait as compared to the plant prior to modulation.

9. Use of a substance which down-regulates the expression of an SRD polypeptide to increase the diameter of starch granules in potato plant leaves, to increase the content of short chains in starch and to reduce the content of long chain parts in starch; the agent that down-regulates expression of an SRD polypeptide is an interfering molecule that interferes with expression of the SRD polypeptide.

10. The use of claim 9, wherein the interfering molecule comprises a structure of formula (I):

Seq_{forward direction}-X-Seq_{Reverse direction}A compound of the formula (I),

in the formula (I), the compound is shown in the specification,

11. The use according to claim 10 wherein the interfering molecule which interferes with the expression of the SRD polypeptide is targeted to position 741-1193 or a transcript thereof of a gene encoding the SRD polypeptide.

12. Use according to claim 9 or 10, wherein the potato plant comprises: cassava, sweet potato, yam, taro, kudzu root, konjak, jerusalem artichoke, yacon and the like.

13. An interfering molecule which down-regulates SRD polypeptide expression and thereby modulates starch traits in the leaves of a potato plant, comprising the structure of formula (I):

Seq_{forward direction}-X-Seq_{Reverse direction}A compound of the formula (I),

in the formula (I), the compound is shown in the specification,

14. A vector comprising the interfering molecule of claim 13.

15. Use of an SRD polypeptide or a gene encoding the same as a molecular marker for identifying starch traits in leaves of a potato plant; the starch character of the leaf of the potato plant comprises: the diameter of the starch granule, and the content of short-chain and medium-long-chain parts in the starch.