Vector Construction And Transformation Of Nitrogen Absorption And Utilization Genes In Rice Based On CRISPR/Cas9 System

Since twenty-first Century,China has gradually developed into a new economic,industrial and agricultural country.However,the aging of population,the deterioration of environment and the shrinking of cultivated land make it very important to improve crop yield.Nitrogen is one of the most important nutritional elements in crop growth and development.It is closely related to the yield of rice.Therefore,it is very necessary to develop and research rice related genes for nitrogen uptake and utilization to ensure stable and high yield of rice.As one of the main grain crops in China,the importance of nitrogen in rice breeding is understandable.A large number of studies have shown that there are many genes related to nitrogen uptake and utilization in rice.NRT(Nitrate-protein symporter)and NLP(Nin-like family protein),The two gene families are involved in the research of nitrogen uptake and utilization in rice.In addition,CRISPR/Cas9 technology as a new gene editing technology has been widely used in gene function research because of its low cost,high efficiency and easy operation.In this paper,we use CRISPR /Cas9 technology to construct knockout vectors of NRT and NLP gene families,and target gene knockout by using Agrobacterium mediated genetic transformation with japonica rice Zhong Hua 11(ZH11)as receptor,so as to get target gene knockout mutants.First,according to the sequence information of NRT family and NLP family gene in rice database,we constructed PYL-CRISPR/Cas9-ubi-NRT/NLP CRISPR target double expression vector with specific target sequence and hygromycin resistance.In addition,Agrobacterium mediated transformation of Japonica Rice Zhonghua 11 was carried out.After screening with hygromycin medium,hygromycin positive PCR was identified.The main results are follows:(1)we successfully constructed CRISPR /Cas9 single gene double target expression vector with specific target sequences and hygromycin resistance,a total of 28,including 15 NRT family genes and 13 NLP family genes.(2)There are 10 genes were successfully obtained the T0 generation of transgenic plants.Which are:1.7,0390,1442,3710,4764,NLP2,1236,1629,3771,2719.(3)After the identification of the hygromycin positive PCR,249 of the 309 T0 transgenic plants were positive,and the positive rate reached 76.85%.(4)After further knockout,there were 5 positive plants near the target site of 0390 gene in the NLP family,and the mutation of base substitution,deletion and insertion occurred in the T0 generation.