Construction And Analysis Of CRISPR/Cas9 Vector About Rice Heading Date Hd1 Gene

Rice(Oryza sativa L.) is one of the most important food crops. Heading date is one of the most important actors, which influence the planting and producing of rice, and decides the ecological zones and regional adaptability of rice. Heading date of rice is a quantitative trait controlled by multiple genes, and it is influenced by itself genetic factors and environmental factors. Hdl(Heading date 1) is one of heading date genes, which is an orthologous gene of Arabidopsis thalina CONSTANS (CO). CO is promtiong flowering, but Hdl has a dual function:to promote heading date under short day conditions, to delay heading date under long day conditions. The CRISPR/Cas system is a popular gene editing technology in recent years, it is that the use of RNA to guide Cas9 nucleases to cut and modify at targeted sites. Compared with the ZFT、 TALEN, CRISPR/Cas system is easier to operate, higher efficiency of mutations, and easier to obtain homozygous in the first generation, and may introduce multiple mutations simultaneously at different sites.CRISPR/Cas9 belongs to TypeⅡ, is a component of a relatively simple system. In this study, firstly construct the vector of CRISPR/Cas9 to knock out Hdl, then introduce to early maturity rice varieties Kitaake and Zhenshan97 to obtain hdl mutants. The effects of CRISPR/Cas9 system are identified by sequecing the hdl mutants, the ways of mutaions, and observing the heading date sooner or later in the filed.In this experiment, using designed the vector of CRISPR/Cas9 knocks out rice Hdl. Through Agrobacterium-mediated transformation method, we introudece the expression vector pCAMBIA 1300::Hd1sgRNA::CRISPR/Cas9 into Kitaake and Zhenshan 97 to obtain hdl mutants. The main results are as follows:1. For the sake of target site specially, according to the Gramene about gene Hdl sequences of japonica rice, indica rice and the sequences of Kitaake, ZS97 which are sequenced, we decide to design a CRISPR/Cas9 target site in the first exon of Hdl. Besides we choose the target site contains an ApaLI restriction site, and consistent with the demand of CRISPR/Cas9 system sgRNA primers.2. The designed Hd1sgRNA primers annealed to double strands and then connect with the vector of the CRISPR/Cas9. Next, connect the Hd1sgRNA::Cas9 with the expression vector pCAMBIA1300. Last, using Agrobacterium-mediated transformation method, we introduced the pCAMBIA1300::Hd1sgRNA::Cas9 into Kitaake and ZS97. After Hygromycin resistance selection and calli differentiation, we obtained 127 regeneration plants of Kitaake background.3. After PCR amplication of Hygromycin gene fragment of the transgenic plants, we obtained 24 Hygromycin positive transgenic plants.4. Determine the mutation in the transgenic plants. Firstly, use PCR amplified Hdl fragment contained the CRISPR/Cas9 target site. Secondly, ApaLI cleaved the amplified products, and marked the undigested products. Lastly, the undigested bands from RFLP analysis were cloned and sequenced We found that 3 out of the 24 rice TO transgenic plants are mutants.Through the comparison of the results of sequences found the way of mutations.5. Analyse the Hdl amplified fragments of the three mutant plants.There were small insertions or deletions or subsitution in one single transgenic plant in the target site. The plant 1-4 is heterozyote, contained a substitution f a base.The Plant2-7 and Plant4-4 are chimeras, have the same way of mutation:there have two kinds of way: one is 3 insertions,another is the deletion of 52 bases.6. The identification of phenotypes of the three mutants in the field, namely the heading date is sooner or later. Compared with the heading date of the Kitaake, Plant1-4 had no significant difference; Plant2-7 and Plant4-4 had a big difference: their heading date is later.