| With the continuous development of urbanization in China,the shortage of rural labor force leads to the high cost of hybrid rice seed production.Therefore,the realization of mechanization of hybrid rice seed production is the fundamental way for the development of hybrid rice industry.Although a number of mechanized hybrid rice seed production technology systems have been developed such as the system based on different grain shape,grain color and herbicide sensitivity between parents.However,it has not yet achieved large-scale industrialization.In this study,we designed a hybrid rice mechanized seed production technology scheme based on plastid genetic markers constructed by chloroplast transformation.Genome editing technology was apply to improve the chloroplast transformation efficiency rate.And fluorescent marker genes or herbicide sensitive genes were transferred into the chloroplast of rice restorer lines to establish a hybrid rice mechanized seed production system with transgenic biological safety.At the same time,under the condition that the chloroplast transformation efficiency is difficult to reach the expected level,this study also designed an experimental scheme to apply herbicide sensitive genes to intelligent female sterile lines to solve the problem of incomplete abortion of female sterile lines.POLLEN TUBE BLOCKED 1(PTB1)was related to pollen tube development.Mutation of cytochrome P450 monooxygenase(Bel)can make plants sensitive to bentazone.And betaine aldehyde dehydrogenase(Badh2)was related to the metabolism of aroma substances in rice.The above three genes were simultaneously knocked out by genome editing technology to quickly create herbicide sensitive intelligent female sterile lines and establish a practical hybrid rice seed production technology system based on female sterility.The main results are as follows:(1)The gene editing technology,which is composed of lustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated protein 9(Cas9),was applied to the tobacco chloroplast transformation system to improve the recombination efficiency by site-specific splicing of chloroplast genome.It was found that both transient and stable expression of gene editing system in chloroplast could effectively improve the efficiency of plastid transformation.We have constructed a CRISPR/Cas9 system vector targeting the intermediate sequence of the homologous recombination fragment of tobacco chloroplast transformation.The gene editing system was applied to tobacco chloroplast transformation in two forms.One way is to co-transform with tobacco chloroplast transformation vector.The foreign genes will not be integrated into the chloroplast genome and will be transiently expressed in the chloroplast.The other way is to construct into tobacco chloroplast transformation vector and integrate it into tobacco chloroplast genome through chloroplast transformation.The foreign genes will express stably in chloroplast.The results showed that the transient expression of CRISPR/Cas9 system in chloroplast could increase the chloroplast transformation efficiency by 6.74 times,and the stable expression could increase 10.82 times.(2)The rice chloroplast transformation vector containing CRISPR/Cas9 system,and the vector was transformed into rice callus by gene gun method.Three rice chloroplast transgenic seedlings were obtained.Firstly,we integrated the gene editing system into the chloroplast transformation vector,and the target sequence was homologous recombination site of rice chloroplast transformation.The normal expression of hpt,e GFP and Cas9 driven by prokaryotic promoter prrn in prokaryotic system of E.coli was verified.Secondly,the effects of six hormones on rice callus proliferation and redifferentiation were compared.IBA and TDZ were selected to carry out orthogonal experiment with two factors and five levels to optimize the hormone ratio in rice differentiation medium.The results showed that the callus redifferentiation efficiency was the highest on the differentiation medium containing 2.5 mg/L IBA and 0.5 mg/L TDZ.The optimization of differentiation medium provided suitable receptor materials for rice chloroplast transformation.Finally,the rice chloroplast transformation vector was introduced into rice chloroplast by gene gun,and three resistant calli were obtained through resistance screening.After several rounds of screening,the resistant calli were transferred into differentiation medium and differentiated into resistant seedlings.The existence of foreign gene fragments was verified by PCR molecular detection,and the transgenic plants were confirmed.However,the transformation efficiency of the system was only 0.25%,and each transgenic line needed 33.33 bombardments.Moreover,due to the low degree of homogenization,the foreign sequence can not be stably inherited to offspring without screening pressure.(3)Taking excellent restorer lines as receptor material,the homozygous mutants of PTB1,Bel and Badh2 genes were obtained by using CRISPR /Cas9 technology.The experimental results showed that the mutants were female sterile,bentazone sensitive and aromatic,which was suitable for mechanized seed production of hybrid rice based on female sterility.Firstly,a multi-target gene editing vector was constructed for five target sites of three genes(Badh2 has only one target site).The transgenic plants derived from Minghui 86,Huazhan,Zhen8 b,Yazhan and R278 were obtained by Agrobacterium mediated genetic transformation.The mutation rates of those three genes were 82.35%,76.47% and 94.11% respectively,which indicated that the double target gene editing system had high mutation efficiency.Different types of mutants such as deletion mutation,base insertion and long segment deletion were obtained by molecular detection.And the homozygous mutants without transgenic fragment were obtained by self breeding of transgenic plants.The results of fertility identification experiments showed that the rice panicle was erect,the seed setting rate was very low,and the pollen fertility of the plant was normal.The mutant plants were female sterile.The bentazone spraying experiment showed that the mutant was very sensitive to this herbicide.The concentration of 2400 mg/L aqueous solution could kill rice seedlings.In addition,the leaves and seeds of the mutants had obvious fragrance,and the content of 2-AP in the seeds was significantly higher than control.(4)Three linked expression cassettes of PTB1 fertility restoring gene,pollen lethal gene expression box and red fluorescent marker gene expression box of intelligent female sterile line were introduced into PTB1,Bel and Badh2 mutants by cross breeding,and bentazon sensitive intelligent female sterile line was obtained.We used mutant as male parent to be hybridized with transgenic plant containing the linkage expression cassette.The bentazon sensitive intelligent female sterile lines of Minghui86,Huazhan,Zhen8 b,Yazhan and R278 were obtained by fluorescence screening and molecular detection and self breeding.This technology system can quickly reform the excellent restorer line into bentazon sensitive intelligent female sterile lines.The non-transgenic seeds separated from the self breeding seeds can be used as the restorer lines in mechanized seed production.The non hybrid seedlings from selfing of male parents can be removed by spraying bentazone on the hybrid rice seedlings.the problem of incomplete abortion of female sterile lines can be solved and the purity of seed production can be guaranteed. |
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