Application of SPL18 gene in enhancing plant yield
A gene and plant technology, applied in the application field of SPL18 gene, can solve the problem of unknown function of OsSPL gene
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Embodiment 1
[0039] Example 1 Construction of Rice SPL18 Gene OsSPL18 Overexpression Vector
[0040] OsSPL18 gene expression box genome sequence (nucleotide sequence shown in SEQIDNO.5) acquisition: design PCR primers:
[0041] OsSPL18-geno-F (5'TGGTACCGATCTCACGTAAATACAATTCTCC) and OsSPL18-geno-R (5'CGGGTACCAATGATATATTTCTGCATAAGTTT) were amplified by PCR using the commercial rice variety Xiushui-134 genome as a template, including the putative OsSPL18 gene promoter, expression sequence and termination The size of the subsequence is a genome fragment of 7.4kb, and the sequence is shown in SEQ ID NO:5. The PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 7.5 minutes, repeating 33 cycles; and then 72°C for 10 minutes.
[0042] Construction of the Agrobacterium T-DNA vector: the binary vector pCambia1300-p35S-G10 vector (InternationalPat.No.PCT / CN2012 / 087069:SEQIDNO.47) contains a glyphosate-resistant gene (EPSPS) (as a marker gene for trans...
Embodiment 2
[0043] The construction of embodiment 2 rice SPL18 gene cDNA overexpression vector
[0044] 1. Cloning of cDNA
[0045] Cloning of rice OsSPL18 gene cDNA: PCR primers OsSPL18-CF (5'GGATCCAACAATGGATTGGGATCTCAAGATG) and OsSPL18-CR (5'GAGCTCCTACTGCCACGAGAATGGGAGCG) were designed, using the cDNA reverse-transcribed from the total RNA of inbred line 9311 as a template, and obtained by PCR amplification cDNA sequence of OsSPL18. The PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 58°C for 15 seconds, 72°C for 90 seconds, repeating 33 cycles; and then 72°C for 10 minutes. The obtained PCR product of about 1.5 kb was cloned into the T-vector pMD19. Then, the corresponding cDNA (SEQ ID NO: 6) was obtained by double digestion with BamHI and SacI, and DNA sequence determination showed that the nucleotide sequence of the cDNA was correct. Then use pCambia1300 as a transition vector, double digest with BamHI and SacI to obtain the sequenced correct OsSPL18cDNA fra...
Embodiment 3
[0063] Embodiment 3, transformation of rice
[0064] The method of obtaining transgenic rice is to adopt the existing technology (Lu Xiongbin, Gong Zuxun (1998) Life Science 10: 125-131; Liu Fan et al. (2003) Molecular Plant Breeding 1: 108-115). The mature and plump seeds of "Xiushui-134" were dehulled, and callus was induced as transformation materials. Take the vectors constructed in Example 1 and Example 2 (pCambia1300-pOsSPL18-OsSPL18-p35S-pZmUbi-1174(geno);
[0065] pCambia1300-p35S-OsSPL18-pZmUbi-1174;
[0066] pCambia1300-pOsSPL18-OsSPL18-ter-p35S-pZmUbi-1174,
[0067] pCambia1300-pOsGA20ox1-OsSPL18-ter-p35S-pZmUbi-1174;
[0068] pCambia1300-pOsTEL-OsSPL18-ter-p35S-pZmUbi-1174;
[0069] Agrobacterium plating of pCambia1300-pOsARGOS-OsSPL18-ter-p35S-pZmUbi-1174). Pick a single colony to inoculate and prepare Agrobacterium for transformation. The callus to be transformed is put into the Agrobacterium bacterium liquid that OD is about 0.6 (preparation of Agrobacteri...
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