Biological Function Research Of BrCRY2b In Chinese Cabbage

Chinese cabbage is one of the vegetables with the largest cultivated area in China.Its yield is related to the market supply and price of vegetables and plays a crucial role in stabilizing people's lives.The cultivation of Chinese cabbage has a long history resulting to numerous variation types.Some excellent agronomic characters have been selected manually for a long time,however,less research have been done on the molecular mechanism of some important agronomic characters of Chinese cabbage and the application of molecular biological technology in the improvement and optimization of Chinese cabbage varieties is far less than that in other crops.The research on the regulation mechanism of hypocotyls elongation of Chinese cabbage is helpful to cultivate new varieties with good characters by genetic engineering technologies,enhance its ability of resistance to lodging and finally achieve the goal of increasing the yield of Chinese cabbage with the help of reasonable cultivation and management technology of Chinese cabbage.The hypocotyl lengths of three Chinese cabbage varieties"Chengyangqing","Zhangqiu 668"and"663"were significantly different under blue light induction.The genes caused this trait were screened and their biological functions were studied.And the following conclusions are obtained:1.Cryptochrome2?CRY2?is responsible for the difference of hypocotyl length between"Chengyangqing"and"663"under blue light.It is found that there was a single base insertion on the third exon of Br CRY2b of"663"causing the frameshift mutation of the protein.The F₁generation of"Chengyangqing×663"represented long hypocotyls after blue light induced,and its F₂groups had a hypocotyls length separation ratio as 3:1,indicating that the trait was a quality trait controlled by a single gene.Crispr/Cas9 and over expression vectors of Br CRY2b were constructed to transform"Guangdongzao".Transgenic plants?T₁?over-expressed Br CRY2b were identified and were verified at DNA,RNA,and protein levels.The phenotype of T₂generation of the transgenic lines was proved that Br CRY2b has the function of regulating hypocotyl elongation of Chinese cabbage under blue light2.Study on the identification of differential genes between"Chengyangqing"and"Zhangqiu 668".Sequencing results showed that there were no significant differences in CRYs between these two lines.It was found that under blue light induction,F₁generation of"Chengyangqing×Zhangqiu 668"also represented a long hypocotyl character,while F₂groups showed continuous change of hypocotyl length closing to normal distribution.The results indicating that the trait was a quantitative character controlled by multiple genes.Using the method of BSA,the differential genes were located to 6 candidate areas distributing on A03,A08 and A09 chromosomes,and the fine mapping of the differential genes is in progress.3.Br CB5 gene family was found to participate in the Br CRY2b mediated blue light signal pathway during the biological function research of Br CRY2b.In order to better understand the roles of Br CB5 in the blue light signaling pathway,bioinformatics analysis was carried out,and we also analyzed the expression patterns of Br CB5s among different tissues and under treatments of salt stress?Na Cl?,osmotic stress(PEG₆₀₀₀),temperature stress?4?,35??,plant hormones?ABA,SA,GA3?stresses at 3 h and 24 h.The results showed that there were various expression patterns of Br CB5s in different tissues and in response to the tested stresses,indicating that Br CB5s played a role in many signaling pathways during plant growth and development.4."663",the Br CRY2b deficient mutant,showed a susceptible phenotype to Hyaloperonospora brassicae?H.brassicae?.In order to find the signal pathway where Br CB5s and Br CRY2b crosstalked,this study focused on downy mildew caused by H.brassicae,and the RNA-seq of the two genotypes,resistant line?R,Chengyangqing?and sensitive line?S,663?,was carried out.DEGs were screened out between the control group and the experimental group of the two genotypes?R-CK vs.R-T,S-CK vs.S-T?,respectively,and GO and KEGG analysis were carried out on these DEGs.It was found that the DEGs of S genotype was enriched in"plant pathogen interaction pathway",DEGs among which participated in MAPK cascade reaction,Ca²⁺signaling,SA and ET signaling pathways.The results also showed that Br CRY2b may be related to the basic defense mechanism of Chinese cabbage against the pathogen,while most of the Br CB5s were related to basic defense mechanism and systemic acquired resistance of Chinese cabbage to downy mildew.