CN107916267A - A kind of high amylopectin starch biosynthesis recombination CBI and its application - Google Patents

Abstract

The invention discloses a high amylopectin biosynthesis recombinant gene CBI and its application. Its nucleotide sequence is shown in SEQ ID NO: 1. A recombinant gene that can inhibit amylose synthesis and enhance starch content can be obtained, providing a A high amylopectin biosynthesis recombinant gene CBI and its application, the present invention is isolated from Escherichia coli JM109‑3 mutant strain and obtained the glgC55 encoding gene of adenosine diphosphate glucose pyrophosphorylase, obtained from potato and combined with starch granules Starch synthase encoding gene GBSSI cDNA and its promoter DNA.

Description

A high amylopectin biosynthesis recombinant gene CBI and its application

technical field

The invention belongs to the technical field of biomedicine and discloses a high amylopectin biosynthesis recombinant gene CBI and its application.

Background technique

Starch is one of the most common and cheapest photopolymerizable carriers next to cellulose. Starch has a single composition, is easy to store, and has a wide range of uses. It is an important industrial raw material for building materials, textiles, papermaking, and oil extraction. In particular, the chemical energy stored in it is easy to convert and utilize again. For this reason, a new understanding of starch at a strategic level is in the ascendant.

Starch comes from plant seeds (such as rice, wheat, corn, etc.), tubers (such as potatoes, etc.), etc. Due to food composition and starch characteristics, potato starch is a widely accepted and used industrial raw material; the starch yield of potato production areas in my country (The amount of tubers required to produce per kilogram of starch) is 1/(7-8), while in developed countries it is 1/(3-4), and the production efficiency is low. The reason is that the starch content in my country's production areas is only 15%-18%. .

Breeding is the basic way to improve or obtain varieties. It is based on the genetic principles of Mendel and Morgan. Hybridization is the basic way to achieve trait-determined gene recombination. In European and American potato crossbreeding, more than 80% of the currently widely planted varieties have been obtained. , wherein the starch content of the varieties used for starch processing purpose is more than 20%.

In Western Europe, the Scottish Agricultural Science Advisory Agency (Edinburgh) is responsible for maintaining the European potato variety database, which collects and records in detail the relevant patents of varieties obtained by hybrid breeding; HZPC, Jalving, Averis, etc. are the main breeding companies in Western Europe. The created potato varieties Agria, Desiree, Bintje, Lady Rosetta, etc. are widely cultivated.

The Russet series of varieties jointly bred by North America are widely used, and the resulting patents include Pat. .5,510,253, Pat.No.5,304,730, Pat.No.5,382,429, Pat.No.5,503,999, Pat.No.5,648,249, Pat.No.5,312,912, Pat.No.5,498,533, Pat.No.5,276,268, Pat.No.4,900,676 , Pat. No. 5,633,434, Pat. No. 4,970,168.

In recent years, driven by the huge demand for starch in the international market, high-quality starch with high flour extraction rate and high viscosity has become even more popular. In the past ten years, international chemical giants, such as Avbe in the Netherlands and BASF in Germany, have invested in starch quality improvement. The breeding research in China has achieved many breeding results, among which the potato variety Amflora improved by genetic engineering is obtained.

The amylopectin content in Amflora tuber starch is more than 95%, and the starch processed by Amflora only needs one-fifth of the amount of traditional potato starch to achieve the same use effect; under the pressure of naturalists, the European Union has BASF has been resisting the planting of genetically modified crops in Western Europe, but in view of the huge temptation of Amflora starch, BASF was authorized by the European Union in 2010 to plant Amflora in Europe.

Starch is not only a component of food for human survival, but fuel ethanol produced from starch is becoming one of the power sources. Therefore, starch is also a component of energy for human activities. In view of people's high expectations for the energy contained in starch, the future will be The huge demand for starch is obvious. Significantly increasing the accumulation of starch in plants has been and is a major issue facing mankind in the past and today.

Starch is composed of two types of glucoside polymers, one is amylose in which glucose alfa-1,4 linear links account for the vast majority, and the other is amylose with multiple alfa-1,6 branch links in the alfa-1,4 linking chain Amylopectin; the main enzymes directly involved in the biosynthesis of starch are: adenosine diphosphate glucose pyrophosphorylase (AGPase.EC 2.7.7.27)), soluble starch synthase (SSS), and starch synthase combined with starch granules (GBSS), starch branching enzyme (BE), starch debranching enzyme (DBE).

Bacterial AGPase is a homotetramer encoded by the glgC gene, and AGPase in higher plants is a heterotetramer (heterologous four-subunit complex), with two identical large subunits and two identical small subunits, The small subunit (SS) performs the metabolic function of AGPase, the large subunit (LS) mainly regulates the activity of AGPase, and the heterogeneous regulation induced by allosteric factors is the main form of regulation of AGPase.

When the single subunit of the bacterial AGPase homotetramer and the small subunit of the plant AGPase heterotetramer exist in an oligomer state, the metabolic group at the N-terminus performs the function of ADP-glucose synthesis, that is, the phosphoric acid Glucose and ATP are used as substrates to synthesize ADP-glucose, which is the substrate for alfa-1,4 or alfa-1,6 glycoside chain extension.

The AGPase subunit G336D mutation in the E. coli K12 mutant strain 618 increased its AGPase activity by 33%. Under the drive of the 35S promoter, the AGPase coding gene glgC16 of the 618 strain was expressed. In potatoes, the AGPase activity and tuber starch content increased by more than 30%, and G336D was expressed Mutating glgC in cassava can increase its AGPase activity by 70% and its root starch content by more than 2 times.

Studies have shown that the plant AGPase small subunit exhibits activity when it exists in the form of an oligomer, and promotes starch synthesis. Activity, inhibiting starch synthesis; many allosteric factors affect the redox state of cysteine 82 in the small subunit of plant AGPase, making it sensitive to regulation.

Bacterial AGPase replaced plant AGPase, no AGPase metabolic subunit dimer formation was observed, and starch synthesis was not inhibited, which is why the expression of glgC16 in potato enhanced the activity of AGPase. Among the AGPase encoding gene transcription, AGPase activity and starch synthesis in plants, the expression level of AGPase and the change of its activity level are highly elastic. Improving plant AGPase activity is the way to increase starch biosynthesis.

However, in further experiments, it was found that the expression of glgC16 in potato plants was driven by the Patatin promoter. Although the AGPase activity in the transformed lines was significantly enhanced, the starch content of tubers was not significantly different from that of the control, that is, the expression results of glgC16 in plants could not be reproduced. The possible reasons include glgC16 itself and the promoter.

At present, changing the ratio of amylose and amylopectin is mainly realized by operating enzymes such as GBSS, SSS, SBE and DBE. Using the antisense RNA technology, the antisense GBSSI is expressed in the potato body, resulting in a decrease or loss of GBSSI activity, which in turn leads to a 70%-100% reduction in amylose content in potato tubers.

In contrast, the expression of the GBSSI gene in amylose-free potato tubers complements the lack of amylose synthesis. The Wx cDNA containing the first intron was reverse-linked to the Wx and maize Adh1 promoters and introduced into rice, the content of amylose in the transgenic rice endosperm decreased, and the endogenous Wx mRNA level in the immature endosperm Also reduced, so that starch quality changes.

Antisense RNA can complement its sense mRNA, so that the encoded protein cannot be synthesized, thereby inhibiting its function. The advantages of antisense RNA are as follows: (1) Antisense RNA inhibits specific gene expression. (2) Low-abundance antisense RNA can also produce high-efficiency repression. (3) Antisense RNA cannot be translated to produce protein, which has high safety. ⑷ The technical operation is simple and easy, and the application range is wide.

GBSSI antisense RNA inhibited the expression of GBSSI, obtained reduced amylose synthesis or only synthesized amylopectin, but did not enhance starch biosynthesis and increase starch accumulation.

Contents of the invention

The purpose of the present invention is to overcome the defects of the above-mentioned technology, obtain a recombinant gene that can inhibit amylose synthesis and enhance starch content, and provide a high amylopectin biosynthesis recombinant gene CBI and its application. The glgC55 gene encoding adenosine diphosphate glucose pyrophosphorylase (GenBank: AY943834) was isolated from the -3 mutant strain, and the starch synthase encoding gene GBSSIcDNA (DQ387451) and its promoter combined with starch granules were isolated from potato DNA.

glgC55 encodes an AGPase subunit (AAY18580) consisting of 431 amino acids, in which there are 4 specific mutations, they are K39E, V71A, I248V and K304E, the former two appear in the Rossmann fold region of the N-terminal metabolic group of the oligomeric subunit The latter two occur within the C-terminal heterogeneously regulated and oligomerized LbH groups of the oligomeric subunits.

The present invention uses glgC55, GBSSIcDNA and its promoter DNA to construct a 35S:glgC::GBSSI:anti-GBSSI recombinant gene, which can be expressed in the target plant to increase starch biosynthesis and increase amylopectin components content.

The present invention has the following features: 1) 35S:glgC::GBSSI:anti-GBSSI is composed of 35S:glgC and GBSSIP:anti-GBSSI; 2) 35S:glgC::GBSSI:anti-GBSSI expressed in plants can make AGPase Vitality increased by 70% compared with the control; 3) 35S:glgC::GBSSI:anti-GBSSI plant expression can reduce potato tuber amylose to 10% (control is 20%); 4) 35S:glgC::GBSSI:anti -GBSSI expression in plants can reduce the regulation of starch biosynthesis by diurnal cycle; 5) 35S:glgC::GBSSI:anti-GBSSI expression in plants can make the starch content of potato tubers 26% higher than that of the control, and the starch viscosity is 35% higher than that of the control %above.

Its specific technical plan is:

A high amylopectin biosynthesis recombinant gene CBI, the nucleotide sequence of which is shown in SEQ ID NO:1.

Application of a high amylopectin biosynthesis recombinant gene CBI in the process of starch biosynthesis.

Compared with prior art, the beneficial effect of the present invention is:

Repeated experiments with different host genotypes in vivo and in vitro showed that the expression of 35S:glgC::GBSSI:anti-GBSSI in plants can simultaneously increase the activity of AGPase, increase the accumulation of starch in storage organs, and increase the viscosity of starch without adding translocation at its end Peptide coding sequence, overcoming the deficiency that only antisense GBSSI expression cannot increase starch accumulation.

Description of drawings

Figure 1 is a schematic diagram of the CBI recombinant gene.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Purpose glgC get

According to the method of Lee et al., the genomic DNA of Escherichia coli JM109 was extracted, and it was used as a template, and glgC-F: 5'-gagttagccatggttaggtttagag-3', glgC-R: 5'- aaagatctctgaacatacatg-3'. is the primer, and PCR is performed under the reaction conditions of pre-denaturation at 95°C for 4min, denaturation at 95°C for 30sec, annealing at 53°C for 45sec, extension at 72°C for 1min, reaction for 30 cycles, final extension at 72°C for 10min, and storage at 4°C. After the PCR product was purified and recovered (Promega; Wizard SV Gel and PCR Clean-Up System), a ligation reaction was established with the recovered product and the pGEM-T vector (promega, pGEM-T Vector Systems, A1360), according to the instructions of the pGEM-T vector Methods: Transform the ligation product into DH5α (Takara company) competent cells, spread the transformed competent cells evenly on the LB solid agar plate containing ampicillin, select white colonies after culturing for 12 hours at 37°C, and inoculate them on the plate containing ampicillin In the liquid LB medium, shake culture for 12 hours. According to the method of "Molecular Cloning Experiment Guide" p19-23, extract the plasmid DNA of the bacterial liquid, wash it thoroughly, and identify it with NcoI and BglII double enzyme digestion. The plasmid contained in the correct single clone is identified as pC-glgC. glgC recombinant bacteria, and the target glgC in pC-glgC was sequenced, and the sequencing results were registered in GenBank with the registration number AY943834.

Acquisition of the GBSSI promoter

According to the method of "Molecular Cloning Experiment Guide" p672-683, CTAB method extracted potato genomic DNA, and used it as a template, using PH-F: 5'-tccttccttttagcagtgtatcaat-3', PH-R: 5'-gaacttgatctttgtgggt-3' as For primers, set the reaction conditions of pre-denaturation at 95°C for 4min, denaturation at 95°C for 30sec, annealing at 53°C for 45sec, extension at 72°C for 1min, reaction for 30 cycles, final extension at 72°C for 10min, and storage at 4°C for PCR. The PCR products were purified and recovered (Promega ; WizardSV Gel and PCR Clean-Up System), use the recovered product and the pGEM-T carrier (promega, pGEM-TVectorSystems, A1360) to establish a ligation reaction, and according to the method of the pGEM-T carrier manual, the ligation product is transformed into DH5α (Takara company ) Competent cells, spread the transformed competent cells evenly on the LB solid agar plate containing ampicillin, select white colonies after culturing for 12 hours at 37°C, inoculate them in the liquid LB medium containing ampicillin, and culture with shaking for 12 hours Hour. According to the method of "Molecular Cloning Experiment Guide" p19-23, extract the plasmid DNA of the bacterial liquid, after fully washing, use the method of "Molecular Cloning Experiment Guide" p237-259, use NcoI and BglII (Takara company) double cut identification, the identification is correct The plasmid contained in the single clone was pG-GBSSIP, and the recombinant bacteria containing pG-GBSSIP were expanded and cultivated, and the target GBSSIP in pG-GBSSIP was sequenced. The correct GBSSIP sequence was registered in GenBank as gene AY948720.

Acquisition of GBSSI cDNA

According to the instructions of the RNA extraction kit (Amresco, K312-50RXN), the total RNA of potato leaves was extracted, using it as a template, HE-F: 5'-gccaaacttcactagacacc-3', HE-R: 5'-ttgatgggtatgagaagcc-3' As primers, RT-PCR was completed with cDNA Synthesis kit (Takara Company, 6130) instructions, and after the PCR product was purified and recovered (Promega; Wizard SV Gel and PCR Clean-Up System), the recovered product and pGEM-T vector (promega, pGEM-T Vector Systems, A1360) to establish the ligation reaction, according to the method of the pGEM-T vector manual, the ligation product was transformed into DH5α (Takara company) competent cells, and the transformed competent cells were evenly spread on LB containing ampicillin On the solid agar plate, after 12 hours of culture at 37, white colonies were selected, inoculated in liquid LB medium containing ampicillin, and cultured with shaking for 12 hours. According to the method of p19-23 in the "Guidelines for Molecular Cloning Experiments", extract the plasmid DNA from the bacterial liquid, wash it thoroughly, and use NcoI and EcoR I to double digest and identify it. The plasmid contained in the correct single clone is identified as pG-GBSSI, and the expanded culture contains pG -GBSSI recombinant bacteria, and the target GBSSI in pG-GBSSI was sequenced, and the sequencing result was registered in GenBank, and its registration number was DQ387451.

Recombinant 35S:glgC obtained

Escherichia coli containing pG-glgC and pC-0380 were cultured in LB liquid medium, and pG-glgC and pC-0380 plasmid DNA were extracted respectively, and enzyme digestion reactions were respectively established according to the method p237-259 of the "Molecular Cloning Experiment Guide", and used Digest pG-glgC with NcoI and BglII, purify and recover the glgC DNA fragment; digest pC-0380 with NcoI and BglII, purify and recover the pC-0380 vector frame DNA fragment; establish the connection according to the instructions of T4 DNA ligase (Takara D2011A) For the reaction, glgC was connected downstream of the 35S promoter; according to the method of "Molecular Cloning Experiment Guide" p976-982, the connection product was transformed into Escherichia coli DH5α (Takara Company) competent cells. Spread the transformed competent cells evenly on the LB solid agar plate containing ampicillin, select colonies after culturing for 12 hours at 37°C, inoculate in the liquid LB medium containing ampicillin, and culture with shaking for 12 hours. According to the method of "Molecular Cloning Experiment Guide" p19-23, extract the plasmid DNA of the bacterial liquid, after fully washing, use the method of "Molecular Cloning Experiment Guide" p237-259, use NcoI and BglII (Takara company) double cut identification, the identification is correct The plasmid contained in the single clone was pC-glgC, and the recombinant bacteria containing pC-GP were expanded and cultured, and the target glgC in pC-glgC was sequenced to obtain the correct sequence pC-glgC, which contained the recombinant gene 35S:glgC.

GBSSIP: anti-GBSSI cDNA construction

Digest pG-GBSSIP and the expression vector pET-28(c) with NcoI and SacI, respectively. The target fragment and the vector were recovered, purified and ligated to obtain the recombinant plasmid pE-Gp, which was digested with PstI and XbaI to obtain about 1100bp Fragment, HindIII single enzyme digestion to obtain a 1000bp fragment. Prove that the GBSSI promoter has been connected to the multiple cloning site MCS of pET-28(c), and the recombinant plasmid pE-Gp.

Extract the plasmid pE-Gp, recover and purify the small fragment after double digestion with PstI and XbaI, about 1.1kb. At the same time, pC-glgC plasmid DNA was digested with PstI and XbaI to recover a large fragment, about 13kb. T4 ligase was connected for 16 hours and then transformed into Escherichia coli JM109 competent cells. Pick the white colony on the solid agar plate coated with IPTG/X-gal/Kan, extract the plasmid DNA, and use PstI and XbaI to digest the small fragment with about 1.2kb, which is consistent with the size of the target fragment. At the same time, three fragments were identified by digestion with HindIII, the large fragment was equivalent to the size of the vector, and the other two fragments were about 1600bp and 900bp in size, which were in line with the expected results. It was confirmed that the GBSSI promoter was linked between PstI and XbaI of the pC-glgC vector MCS. The resulting recombinant plasmid was named pC-gGp'.

Recombinant gene 35S:glgC::GBSSI:anti-GBSSI obtained

Extract the plasmid DNA of pG-GBSSI and pC-gGp', digest with ScaI and XbaI respectively, recover and purify the target fragment and vector, connect and transform Escherichia coli JM109, pick a single colony, extract the plasmid, double digest with ScaI and XbaI A fragment of about 1000bp was obtained by enzyme digestion, and a fragment of about 2000bp was obtained by single digestion with PstI. It was confirmed that the recombinant plasmid contained glgC initiated by the GBSSI promoter and GBSSI cDNA initiated by the GBSSI promoter, and the recombinant plasmid pCB1 was obtained, which contained For the target gene 35S:glgC::GBSSI:anti-GBSSI, see Figure 1 for details.

Recombinant gene function detection

According to the method of Visser et al., the stem section of sterile potato seedlings with a diameter of 2-4mm was used as explants, and the target gene 35S:glgC and GBSSIP:glgC were transformed into potatoes by the Agrobacterium-mediated pathway; PCR-positive transformation lines were subjected to Southern Hybridization (Beijing Milebo Medical Technology Co., Ltd., Fadigoxigenin Labeling Kit, Digoxigenin Hybridization Detection Kit) to obtain a single-copy insertion line, RT-PCR and Northern hybridization (Beijing Milebo Medical Technology Co., Ltd., Digoxigenin Labeling Kit (Digoxigenin Labeling Kit, Digoxigenin Hybridization Detection Kit) to identify whether the target gene is transcribed, the AGPase activity is measured by the method of Yao et al., the starch content of the tuber is measured with the starch test box (BioAssay company, EnzyChrom Starch Assay Kit), the tuber The content of amylose is carried out according to the method of Hovenkamp-Hermelink et al.; the fully washed and dried starch is made into a 4% (w/v) suspension, and the starch is determined according to the 3mm (Shanghai Shenyi Glass Products Co., Ltd.) capillary viscometer method viscosity.

Through methods such as PCR, 4 mutations in the protein encoded by glgC55 used in the present invention are realized by point mutation under in vitro conditions, and the same DNA sequence of glgC55 is obtained; in addition, the present invention obtains 35S:glgC: The :GBSSI:anti-GBSSI recombinant gene can also obtain the recombinant gene 35S:glgC and GBSSI:glgC by joining with other restriction enzymes.

The above is only a preferred specific embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field within the technical scope disclosed in the present invention can obviously obtain the simplicity of the technical solution. Changes or equivalent replacements all fall within the protection scope of the present invention.

sequence listing

<110> Ningxia University

<120> A high amylopectin biosynthesis recombinant gene CBI and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4067

<212>DNA

<213> Artificial Sequence

<400> 1

agctctccca ctaggtcgac ctgcaggcgg ccgcgaattc actagtgatt gccaaacttc 60

actagacacc aaatcaacct tgtcacagat aggactcagg aaccatactc tgactcacaa 120

tggtttaagg gctgttaaca agcttgatgg gctccaatca agaactaata ctaaggtaac 180

acccaagatg gcatccagaa ctgagaccaa gagacctgga tgctcagcta ccattgtttg 240

tggaaaggga atgaacttga tctttgtggg tactgaggtt ggtccttgga gcaaaactgg 300

tggactaggt gatgttcttg gtggactacc accagccctt gcagcccgcg gacatcgggt 360

aatgacaata tccccccgtt atgaccaata caaagatact tgggatacta gcgttgcggt 420

tgaggtcaaa gttggagaca gcattgaaat tgttcgtttc tttcactgct ataaacgtgg 480

ggttgatcgt gtttttgttg accacccaat gttcttggag aaagtttggg gtaaaactgg 540

ttcaaaaatc tatggcccca aagctggact agattatctg gacaatgaac ttaggttcag 600

cttgttgtgt caagcagccc tagaggcacc taaagttttg aatttgaaca gtagcaacta 660

cttctcagga ccatatggag aggatgttct ctttcattgc caatgattgg cacacagctc 720

tcattccttg ctacttgaag tcaatgtacc agtccagagg aatctatttg aatgccaagg 780

tcgctttctg catccataac attgcctacc aaggccgatt ttctttctct gacttccctc 840

ttctcaatct tcctgatgaa ttcaggggtt cttttgattt cattgatgga tatgagaagc 900

caatcgaatt cccgcggcta ccatggtata tctccttctt aaagttaaac aaaattattt 960

ctagaaataa ttttgtttaa ctttaagaag gagatatacc atggcggccg cgggaattcg 1020

attacccaca aagatcaagt tcactccctt tccacaaaca atggtagctg agcatccagg 1080

tctcttggtc tcagttctgg atgccatctt gggtgttacc ttagtattag ttcttgattg 1140

gagcccatca agcttgttaa cagcccttaa accattgtga gtcagagtat ggttcctgag 1200

tcctatctgt gacaaggttg attggtgtc tagtgaagtt tggcttcttg acacaaagtg 1260

gtgtgaagct gtgatgcttg ccatgtgatg tgtcgtctac aaaaagggga atctaccaga 1320

gaataagcta cagatgaaca agaacaaaac agaaattgat ttctgagaag aagaagaaga 1380

agaggaagca ttcacattta tcaccgatta cacagtaggc aagagaatca aaatcagaat 1440

agatgatatg agatatgaaa caacgtttat acaccataac acgattcata atagaatgta 1500

gggaaacatg catgagatca gaaataatta gaggagatga gtaaaagtta ccacttgttg 1560

agctgtgtga gtgagtgagt gagtgagtga gtgtgagaat gaggaggtgc ctgccttatt 1620

agtagcaggt ttcagtgaca cgtgtcaaga gaataacggg tggctatccc ttagcggaag 1680

gcaactgtgg acactgtatt atagggaaat gctcatcgac agtattgtgg gccctctctt 1740

tgttgattca cggctggact tcaacttggg ccttgcaatg ggcccctccg gttctgtctc 1800

ctagtatcta aaaagctaaa ccaactccct cctaccgcta ccacttgaca ttcctatgtc 1860

tcgtgttaat taaattatta ttatagtaat aaaaaataat atctaatgta ctggtactgg 1920

tccctccact agaattttgt tgcatttttt agtattaaga ttgagatgca tggttctatt 1980

acaaaattga tacctgcagg catgcaagct tggcactggc cgtcgtttta caacgtcgtg 2040

actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 2100

gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 2160

atggcgaatg ctagagcagc ttgagcttgg atcagattgt cgtttcccgc cttcagttta 2220

gcttcatgga gtcaaagatt caaatagagg acctaacaga actcgccgta aagactggcg 2280

aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc gtcaacatgg 2340

tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa 2400

gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc 2460

cagctatctg tcactttat gtgaagatag tggaaaagga aggtggctcc tacaaatgcc 2520

atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag 2580

atggaccccc accacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 2640

agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 2700

cttcgcaaga cccttcctct atncataagg aagttcattt catttggaga gaacacgggg 2760

gactcttgac catggttagt ttagagaaga acgatcactt aatgttggcg cgccagctgc 2820

cattgaaatc tgttgccctg atactggcgg gaggacgtgg tacccgcctg aaggatttaa 2880

ccaatgagcg agcaaaaccg gccgtacact tcggcggtaa gttccgcatt atcgactttg 2940

cgctgtctaa ctgcatcaac tccgggatcc gtcgtatggg cgcgatcacc cagtaccagt 3000

cccacactct ggtgcagcac attcagcgcg gctggtcatt cttcaatgaa gaaatgaacg 3060

agtttgtcga tctgctgcca gcacagcaga gaatgaaagg ggaaaactgg tatcgcggca 3120

ccgcagatgc ggtcacccaa aacctcgaca ttatccgccg ttataaagcg gaatacgtgg 3180

tgatcctggc gggcgaccat atctacaagc aagactactc gcgtatgctt atcgatcacg 3240

tcgaaaaagg cgcacgttgc accgttgctt gtatgccagt accgattgaa gaagcctccg 3300

catttggcgt tatggcggtt gatgagaacg ataaaattat cgaattcgtt gaaaaacctg 3360

ctaacccgcc gtcaatgccg aacgatccga gcaaatctct ggcgagtatg ggtatctacg 3420

tctttgacgc cgactatctg tatgaactgc tggaagaaga cgatcgcgat gagaactcca 3480

gccacgactt tggcaaagat ttgattccca aggtcaccga agccggtctg gcctatgcgc 3540

acccgttccc gctctcttgc gtacaatccg acccggatgc cgagccgtac tggcgcgatg 3600

tgggtacgct ggaagcttac tggaaagcga acctcgatct ggcctctgtg gtgccggaac 3660

tggatatgta cgatcgcaat tggccaattc gcacctacaa tgaatcatta ccgccagcga 3720

aattcgtgca ggatcgctcc ggtagccacg ggatgaccct taactcactg gtttccggcg 3780

gttgtgtgat ctccggttcg gtggtggtgc agtccgttct gttctcgcgc gttcgcgtga 3840

attcattctg caacattgat tccgccgtat tgttaccgga agtatgggta ggtcgctcgt 3900

gccgtctgcg ccgctgcgtc atcgatcgtg cttgtgttat tccggaaggc atggtgattg 3960

gtgaaaacgc agaggaagat gcacgtcgtt tctatcgttc agaagaaggc atcgtgctgg 4020

taacgcgcga aatgctacgg aagttaggc ataaacagga gcgataa 4067

Claims (2)

1. A kind of 1. high amylopectin starch biosynthesis recombination CBI, it is characterised in that its nucleotide sequence such as SEQ ID NO:1 It is shown.
2. 2. applications of the high amylopectin starch biosynthesis recombination CBI during Starch biosynthase described in claim 1.