Cloning and Application of Rice Heading Time Gene haf1
A technology of heading stage and rice, applied in the field of functional verification and application, the isolation and cloning of the gene HAF1, can solve the problems of delayed heading, prolonged growth period, decreased yield, etc.
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Embodiment 1
[0053] Embodiment 1: the isolated clone of HAF1 gene
[0054] 1. Obtaining late heading mutants
[0055]The mutants identified in the present invention are from rice T-DNA insertion mutants (for the creation method of the mutant library, please refer to the paper: Wu et al., Development of enhancer trap lines for functional analysis of therice genome, Plant J (2003): 418-427; Zhang et al., Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing13,804 T-DNA flanking sequences from an enhancer-trap mutant library, Plant J(2007):947-959). The screening and identification method of mutants is as follows: from the above-mentioned rice T-DNA insertion mutant library (see http: / / rmd.ncpgr.cn / ), the T0 generation transgenic rice seeds are planted in the field after conventional seed soaking and germination procedures After getting each T 1 A total of 20 plants were planted in the generation family. The plants were planted in the...
Embodiment 2
[0065] Example 2: Experimental verification of functional complementarity
[0066] 1. Construction of complementary vectors
[0067] The complementary vector constructed in Example 1 is pC2301-HAF1 (see image 3 Figure A in and Figure 8), the constructed vector was electrotransformed into Agrobacterium tumefaciens EHA105 (purchased from CAMBIA, http: / / www.cambia.org / daisy / cambia / materials / overview.html) strain.
[0068] 2. Genetic transformation
[0069] The Agrobacterium-mediated genetic transformation method (Wu et al., Development of enhancer trap lines for functional analysis of the rice genome.2003, Plant J.35:418-427) was used to introduce the complementary strain into the callus of the haf1 mutant. Cultivation, infection, co-cultivation, screening of calli with resistance to G418 (purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.), differentiation, rooting and seedling hardening and transplanting in the field to obtain transgenic plants (Agrobacterium-mediat...
Embodiment 3
[0071] Example 3: HAF1 regulates known heading date related regulatory genes
[0072] In order to analyze the molecular mechanism of HAF1 gene regulating rice flowering, the present invention uses quantitative RT-PCR technology to detect the expression of related heading date regulation genes in wild-type (Zhonghua 11) and haf1 mutant plants. The result is as Figure 4 As shown, the expression rhythm of HAF1 gene gradually increased at night and peaked at night, and gradually increased during the day under long-day light (16 hours of light / 8 hours of darkness) or short-day (8 hours of light / 16 hours of darkness) conditions. decreased and reached a trough in the evening, but in the haf1 mutant, the expression of the HAF1 gene was significantly reduced (such as Figure 4 panels A and B in). By detecting the expression of the circadian clock gene OsGI (LOC_Os01g08700), it was found that there was no difference in the expression of OsGI in the wild-type and haf1 mutants, indicat...
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