Method for cultivating soft rice by using transcriptional activation factor sample effector nuclease technology

A transcription activator and effector technology, applied in the field of soft rice with low amylose content, can solve the problems of large randomness, long time, non-specific mutations, etc.

Inactive Publication Date: 2019-10-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional method of plant breeding is realized by mutagenesis, screening and selfing, which takes a long time, is highly random, and the mutation is not specific, which brings many unfavorable factors to the breeding work.

Method used

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  • Method for cultivating soft rice by using transcriptional activation factor sample effector nuclease technology
  • Method for cultivating soft rice by using transcriptional activation factor sample effector nuclease technology
  • Method for cultivating soft rice by using transcriptional activation factor sample effector nuclease technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Identify rice OsGBSSI Synthesis of the functional module TN12a at the left target site of the gene

[0018] The TN12a module was synthesized by gene synthesis method (Nucleic Acids Research, 2004, 32, e98). The designed primers are:

[0019] TN12a -1 GGATCCCTCACTCCAGCACAGGTGGTTGCGATCGCA TCCA ATATCGGAGG TAAGCAGGCG (shown in SEQ ID NO. 3)

[0020] TN12a -2 CCTGACAAAGGACCGGCAACAGACGCTGCACAGTC T CCAA CGCCTGCTTA CCTCCGATAT (shown in SEQ ID NO.4)

[0021] TN12a -3 GTTGCCGGTCCTTTGTCAGGATCATGGCCTGACGC CAGAT CAGGTAGTCG CAATCGCAAG (shown in SEQ ID NO.5)

[0022] TN12a -4 GCGCTGGACTGTCTCAAGGGCCTGCTTGCCTCCAT TGTTG CTTGCGATTG CGACTACCTG (shown in SEQ ID NO.6)

[0023] TN12a -5 CCCTTGAGACAGTCCAGCGCTTGTTGCCTGTTCTAT GCCA GGATCACGGC CTGACTCCTG (shown in SEQ ID NO. 7)

[0024] TN12a -6 TGTTTACCACCATCGTGAGACGCAATGGCGACCACT TGGTCAGGAGTCAG GCCGTGATCC (shown in SEQ ID NO. 8)

[0025] TN12a -7 TCTCACGATGGTGGTAAACAAGCACTGGAAACGGT TCAAA GACTCCTCCC AGTGCTGTGT (shown in SEQ ID NO.9)

[0026] TN12...

Embodiment 2

[0058] Example 2: Identify rice OsGBSSI Synthesis of the functional module TN12b at the right target site of the gene

[0059] Using gene synthesis method (Nucleic Acids Research, 2004, 32, e98) TN12b. The designed primers are:

[0060] TN12b-1 GGATCCCTCACTCCAGCACAGGTGGTTGCGATCGCA TC CAATATCGGAGGTAAGCAGGCG (shown in SEQ ID NO.41)

[0061] TN12b -2CCTGACAAAGGACCGGCAACAGACGCTGCACAGTCTC CAACGCCTGCTTACCTCCGATAT (shown in SEQID NO.42)

[0062] TN12b-3 GTTGCCGGTCCTTTGTCAGGATCATGGCCTGACGCCA G ATCAGGTAGTCGCAATCGCAAG (shown in SEQ ID NO. 43)

[0063] TN12b-4 GCGCTGGACTGTCTCAAGGGCCTGCTTGCCTCCATTA TTGCTTGCGATTGCGACTACCTG (shown in SEQ ID NO.44)

[0064] TN12b-5 CCCTTGAGACAGTCCAGCGCTTGTTGCCTGTTCTATGC CAGGATCACGGCCTGACTCCTG (shown in SEQ ID NO.45)

[0065] TN12b-6 TGTTTACCACCGATGTTAGACGCAATGGCGACCACTT GGTCAGGAGTCAGGCCGTGATCC (SEQID NO.46)

[0066] TN12b-7TCTAACATCGGTGGTAAACAAGCACTGGAAACGGTTCAAAGACTCCTCCCAGTGCTGTGT (shown in SEQID NO.47)

[0067] TN12b-8 CTATAGCTACGACTTGTTCAGGTGTCAAACCATGATC TTGACACAG...

Embodiment 3

[0099] Example 3: Targeting GBSSI Construction of TALEN Plant Expression Vector of Gene

[0100] The left target site recognition module pUC19 (TN12a) and the right target site recognition module pUC19 (TN12b) of GBSSI gene were double digested with BamHI and SacI. The digested products were separated by Agarose electrophoresis and gel tapping to recover 1500bp fragments. The SFokIA (pUC19) vector and TN12a fragment were ligated with T4 DNA ligase; the SFokIB (pUC19) vector and TN12b fragment were ligated with T4 DNA ligase. The above linkers were transformed into Escherichia coli DH5α, single colonies of Escherichia coli transformants were picked for liquid culture, the plasmids were extracted and the enzyme digestion was carried out, and finally the full sequence analysis and determination of the inserts in the positive plasmids were performed to obtain the targeted GBSSI The two TALEN plasmids of the gene are named SFokIA【TN12a】pUC19 and SFokIB【TN12b】pUC19.

[0101] The SFokIA...

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Abstract

The invention discloses a method for cultivating soft rice by using a transcriptional activation factor sample effector nuclease technology. The method for cultivating the soft rice by using the transcriptional activation factor sample effector nuclease technology comprises the specific steps that according to gene sequence analysis, two segments of specific nucleotide sequences are screened out from a rice starch synthase GBSSI genomic DNA, transcriptional activation factor effector recognition modules of two segments of target site sequences are separately established, and the transcriptional activation factor effector recognition modules are connected with an FokI II nuclease expression unit in series. A plant expression vertor SFokIAB[TN12ab] of the target GBSSI gene TALEN is obtained,and then the expression vector is converted to rice through agrobacterium-mediated transformation. By using the method for cultivating the soft rice by using the transcriptional activation factor sample effector nuclease technology, content of amylose in the rice can be significantly reduced, and thus the superior soft rice is obtained.

Description

Technical field [0001] The invention belongs to the field of plant biotechnology, and specifically uses transcription activator-like effector nuclease technology to directionally shear the rice starch synthase GBSSI gene to cultivate soft rice with low amylose content. Background technique [0002] Starch is the main ingredient in rice, consisting of amylopectin (70%-80%) and amylose (20%-30%). Starch molecules exist in rice in the form of starch granules, and their shapes are mostly irregular polygons. The diameter of rice starch grains is between 3-10 μm. The synthesis of higher plant starch is carried out in plastids, and the formation of starch is accomplished through the crystallization of high molecular weight polymers. High molecular weight polymers include amorphous starch molecules, proteins and lipids. The whole process of starch synthesis is mainly completed by four major enzymes, including ADP (adenosine diphosphate)-glucose pyrophosphorylase, starch synthase, starch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82C12N9/22C12N15/55A01H5/00A01H6/46
CPCC12N9/1051C12N9/22C12N15/8245C12Y204/01021
Inventor 彭日荷姚泉洪田永生高建杰许晶付晓燕李振军韩红娟王波王丽娟张福建黄悠楠张文慧
Owner SHANGHAI ACAD OF AGRI SCI
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