A kind of glw10 gene controlling rice grain length and 1000-grain weight and its encoded protein and application
A thousand-grain weight and gene technology, applied to the GLW10 gene and its encoded protein and application fields, can solve the problem of unclear regulatory network, and achieve the effect of increasing the thousand-grain weight of rice and improving rice yield.
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Embodiment 1
[0028] Example 1 CRISPR / Cas9 Knockout Vector Construction of GLW10
[0029] In the present invention, the CRISPR / Cas vector construction kit of Baige Company is used to construct the knockout vector of GLW10, and the specific process is as follows:
[0030] (1) Use the targetDesign online tool of the http: / / skl.scau.edu.cn / website to design the knockout target site; enter the GLW10 gene nucleotide sequence (SEQ ID NO.2) on the website to generate a 19bp gRNA Target sequence T1, the target sequence is as follows:
[0031] T1: 5'-GGGTGCTTCCTGTCCAAGC-3' (SEQ ID NO.3)
[0032] (2) Enter the above target sequence T1 into the Oligo sequence online design website of Bioge Company (http: / / 121.41.105.238 / index / excrispr), select the BGK03 vector, and generate the Oligo sequence corresponding to the kit, Oligo-F and Oligo The -R sequence is as follows:
[0033] Oligo-F: 5'-TGTGTGGGGTGCTTCCTGTCCAAGC-3' (SEQ ID NO.4)
[0034] Oligo-R: 5'-AAACGCTTGGACAGGAAGCACCCCCA-3' (SEQ ID NO.5)
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Embodiment 2
[0045] Example 2 Construction of GLW10 overexpression vector
[0046]500 ng of total RNA was used for reverse transcription to synthesize cDNA using the reverse transcription kit from Takara Company and operating according to the instructions. Using the synthesized cDNA as a template to amplify the coding region sequence (SEQ ID NO.1) of the GLW10 gene, the amplification primers F and R respectively carry KpnI and BamHI restriction sites (sequences shown underlined), and the sequences are as follows:
[0047] F: 5'-CGG GGTACC ATGGGGTGCTTCCTGTCCA-3' (SEQ ID NO. 6)
[0048] R: 5'-CGC GGATCC ACCTCGCCAGCTATTTTG-3' (SEQ ID NO.7)
[0049] The GLW10 gene coding region sequence amplified above was connected between the KpnⅠ and BamHI restriction sites of the plant expression vector pCAMBIA2300-35S-eGFP using T4 ligase to obtain the overexpression vector pCAMBIA2300-35S-GLW10- eGFP (see figure 2 ).
Embodiment 3
[0050] Example 3 Transformation of rice Nipponbare with GLW10 knockout vector and overexpression vector
[0051] (1) Transform the plasmids of the GLW10 knockout vector GLW10-BGK03 constructed above and the overexpression vector pCAMBIA2300-35S-GLW10-eGFP into Agrobacterium EHA105
[0052] The specific process of the Agrobacterium transformation method is as follows: take out the EHA105 competent cells from the -80°C refrigerator, and thaw quickly; add 1 μL of plasmid to a tube of competent cells, and place on ice for 30 minutes; freeze in liquid nitrogen for 2 minutes; 37 ℃ water bath for 5 minutes to thaw the cells; then immediately add 5 times the volume of LB medium without anti-antibody, and culture at 28 ℃ and 170rpm for 2-3 hours on a shaker; centrifuge at 7000rpm for 2 minutes, resuspend the cells in a volume of 100 μL of LB medium; then spread on LB plates containing rifampicin and kanamycin resistance, blow dry, and culture at 28°C for 2-3 days.
[0053] (2) Transfo...
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