Study On The Function Of BrGID1a In Regulating The Development Of Leaf Head Size In Chinese Cabbage

Chinese cabbage(Brssica rapa L.ssp.pekinensis)originated in China,also known as Chinese cabbage.For a long time,the sown area of Chinese cabbage has been maintained at about 30 million mu,which is one of the vegetable crops with the largest coverage and the highest yield in China.Therefore,it has the title of "national cuisine",and its abundance and apology also play a key role in stabilizing people’s daily life.Leaf head is the main edible part of Chinese cabbage.The size of leaf head has become one of the important agronomic characters of Chinese cabbage.But so far,there are still some problems about the molecular regulation mechanism of regulating the size and development of Chinese cabbage leaf head.Therefore,mining the important genes regulating the size and development of Chinese cabbage leaf head has become the key to innovate Chinese Cabbage Germplasm by using molecular breeding technology(including gene editing,transgenic,molecular marker assisted selection,etc.).In order to achieve the above purpose,this study first used two Chinese cabbage inbred lines Y2(large leaf head)and Y7(small leaf head)with significant difference in leaf head size as materials to preliminarily locate the locus controlling leaf head size of Chinese cabbage through BSA technology,and then combined with the GWAS results in the early stage of the laboratory,located a key gene BrGID1a on chromosome A05 that may be involved in the regulation of leaf head size of Chinese cabbage,The function and mechanism of BrGID1a involved in the development of leaf head size of Chinese cabbage were further analyzed by means of molecular biology.The main results are as follows:1.F2 population was constructed with two Chinese cabbage inbred lines Y2(large leaf head)and Y7(small leaf head)with significant difference in leaf head size as parents,and then the leaf head size and other traits of F2 population composed of 600 single plants were investigated.The results showed that the gross weight and head weight of F2 population showed obvious normal distribution,which showed that leaf head size was a quantitative trait controlled by multiple genes.Further,different individual plants with significant size differences were selected from F2 population to construct the liBrry,and then the genetic loci controlling the traits related to leaf bulb size were located by BSA method.Through relevant bioinformatics analysis,they were located on chromosome A05,about 4.1Mb between 20467001 and 24574670.2.Through the collaborative analysis of BSA localization results and GWAS localization results in the early stage of the laboratory,a key candidate gene Br039490 that may participate in the development of leaf head size of Chinese cabbage was screened.This gene encodes an gibberellin receptor protein,which is evolutionarily homologous with Arabidopsis GIDla.Therefore,this gene is named BrGID1a.3.Using Y2 and Y7 as materials,the CDS sequence and gene sequence of BrGID1a gene were cloned and sequenced.The results showed that there was no difference in the promoter sequence of BrGID1a gene(2000 bp upstream of ATG)between Y2 and Y7,but BrGID1a-Y7 gene in the coding region was terminated early,resulting in great differences in amino acid sequence,protein secondary structure and conserved motif between BrGID1a-Y7 and BrGID1a-Y2.4.Specific primers were designed according to the sequence differences between Y2 and Y7 of BrGID1a gene,which could amplify BrGID1a-Y7 but could not amplify BrGID1a-Y2.Using the primers validated in different groups found that small plant of individuals in the F2 populations can be amplified to the parents in the same size in Y7 stripe,but in some of the single plant with larger stripe can swell to purpose,others cannot,for can swell to the individual further validation of stripe found they were chimeras.This result indicated that the sequence differences of BrGID1a-Y7 and BrGID1a-Y2 genes could be used as molecular markers to better distinguish the size of leaf head of Chinese cabbage.In addition,more than 200 commodities were used as materials for further verification of this marker,and it was found that the varieties carrying the homozygous BrGID1a-Y7 gene generally had small leaf heads,but the varieties with small leaf heads did not necessarily carry BrGID1a-Y7 gene,which also indicated that although BrGID1a played an important role in regulating the development of Chinese cabbage leaf head size,But there should be other genes involved in the process,a trait controlled by multiple genes.5.To further verify the role of BrGID1a-Y7 and BrGID1a-Y2 genes in regulating the size of plant organs,in this study,we constructed a plant overexpression vector driven by CAMV-35S promoter and overexpressed in Arabidopsis thaliana.The results showed that over-expression of BrGID1a-Y2 gene in Arabidopsis thaliana could significantly promote the growth of transgenic plants.The over-expression of BrGID1a-Y7 gene had no significant effect on the growth of transgenic plants.6.In order to understand the mechanism of functional differences between BrGID1a-Y7 and BrGID1a-Y2,the yeast two-hybrid method was used to study the interaction between BrGID1a-Y7 and BrGID1a-Y2 with DELLA protein in Chinese cabbage.The results showed that BrGID1a-Y7 could only interact with BrRGL3-Y7,but BrGID1a-Y2 could not interact with any Chinese cabbage RGL3 protein.Therefore,we speculate that this may be the key to the functional difference between the two,but the specific reasons need further research.In conclusion,through this study,we obtained a key regulatory gene that may be involved in the development of Chinese cabbage leaf head size and developed a closely linked molecular marker.It lays a foundation for breeding new Chinese cabbage varieties by molecular assisted breeding in the next step.