Identification,Cloning And Expression Analysis Of Genes Related To Gibberellin Metabolism In Pak Choi

Pak choi(Brassica rapa ssp.Chinensis Makino)belongs to Cruciferae Brassica,which is a representative seed vernalization vegetable.Early bolting often happened in spring production due to low temperature and gibberellin(GA)plays an important role during this process.Therefore,it is of great practical significance to study the mechanism of gibberellin in the process of plant flower transformation.In this experiment,the content of GA in shoot apex of pak choi was measured at different developmental stages.At the same time,to elucidate the molecular mechanisms of these changes in GA content,using chinese cabbage as the reference genome,the expression profile in each period of shoot apices and the expression of homologous genes encoding enzymes directly involved in GA metabolism were analyzed.Based on these results,the GA metabolism genes GA20ox and GA2ox were cloned,and their spatial expression pattern were analyzed and over-expression vector were constructed,which will provide a foundation for their functional verification.The main results were as follows:1.Comparing the gibberellin content in shoot apices during vernalization and defferent developmental stages in pak choi,the results showed that the GA content increased significantly after low-temperature treatment(4℃)and then decreased rapidly with vegetative growth.During floral bud initiation,the GA content increased rapidly until it peaked upon floral bud differentiation stage 1.2.The transcriptom sequencing results in different developmental stages of pak choi were analyzed.The results showed that the percentages of obtained high quality reads from 6 sequencing samples--after low temperature treatment(V0 and CKO),10 d after transplanting(V10),15 d after transplanting(V15)and flower bud differentiation stage 1(V16 and CK16)which can mapped to the reference genome of chinese cabbage were 82.94%,84.80%,84.57%,85.08%,84.25%and 84.77%,respectively.The proportion of mapped genes was high.3.Based on transcriptom sequencing results,the expression of homologous genes encoding enzymes directly involved in GA metabolism was analyzed.The results showed that the changes in the expression of four genes involved in GA synthesis(Bra035120,Bra009868,Bra015394 and Bra013890)were correlated with the changes in GA content,but for the rest of genes,the expression of some genes were irregular,the expression of some gene were not expressed.4.Seven differentially expressed genes(Bra005596,Bra009285,Bra022565,Bra008362,Bra033324,Bra010802,and Bra030500)were identified.The differential expression of these genes were directly correlated with changes in GA content,suggesting that these genes were directly related to vernalization,floral bud initiation and development and could be further studied.These results contribute to the understanding of the molecular mechanism of GA metabolism in pak choi and other chinese cabbage vegetable.5.Arabidopsis homologous genes Bra009285(GA20ox3),Bra010802(GA2ox2)and Bra030500(GA2ox6)was cloned from pak choi by RT-PCR.Their full length of ORF was 1140 bp,999bp and 1002bp,encoding 332,379 and 333 amino acids,respectively.Through multiple alignment and phylogenetic analysis with other homologous protein,these three genes were confirmed as homologue genes of GA20ox3,GA2ox2 and GA2ox6,and be named BrcGA20ox3,BrcGA2ox2 and BrcGA2ox6.6.The expression pattern of BrcGA20ox3,BrcGA2ox6 and BrcGA2ox2 in different developmental stages and in different organs of pak choi were analyzed,the results showed that the expression of the BrcGA20ox3 and BrcGA2ox6 after vernalization was increased sharply,then decreased rapidly,and later was low even no expression.The expression of BrcGA2ox2 after vernalization was decreased,and then was increased a little in the vegetative growth stage and then was reduced.BrcGA20ox3 is mainly expressed in old leaves and young leaves,and was not detected in flower buds and roots almostly;the expression of BrcGA2ox6 was the highest in flowers,old leaves and in stems a little lower;the expression of BrcGA2ox2 was highly in root,then in flower and flower buds.7.7.Based on the genes sequences of BrcGA20ox3,BrcGA2ox6 and BrcGA2ox2,the over-expression vector of these genes were constructed and then the vector were transformed into Agrobacterium GV3101 and LBA4404,which provide a foundation for functional verification of transgenic plants.