Functional Analysis Of Genes Regulating Growth Cone Differentiation In Winter Brassica Rapa

Northern winter Brassica rapa has excellent cold resistance,and is the only type of winter rapeseed that can be wintered in the cold regions of northern China.Studies have found that the botanical characters such as slow-growing,developed root system,concave growth cone and creeping growth at seedling stage are closely related to cold resistance.The results of study have indicated that the varieties with hollow growth cone are much stronger cold resistance than the varieties with raised growth cone,so it is speculated that there may be differences in the regulation mechanism of the growth cone characteristics of varieties with different cold resistance.In this study,six genes related to the regulation of growth cones and significantly different from laboratory transcriptome data were selected,and their expression levels in different cold-resistant winter rapeseed growth cones were analyzed.Two genes with significant differences were selected for functional analysis,the main results are as follows:1.Screened STM and CUC2 genes related to the morphology of growth cones,among them,the relative expression of STM in varieties with strong cold resistance and recessed growth cones was lower than those with weak cold resistance and raised cones.while the relative expression of CUC2 gene was opposite to that of STM gene,and the relative expression in varieties with strong cold resistance and recessed growth cones was higher than that of the varieties with poor cold resistance and raised growth cones.2.STM and CUC2 genes with length of 1554 bp and 1104 bp were cloned.After low temperature stress,the STM and CUC2 genes were up-regulated and reached the highest value at 4 ℃.There were significant differences in the relative expression of STM and CUC2 genes among different cultivars.The relative expression of STM gene in Lenox with raised growth cone was higher than that in Longyou 7 with recessed growth cone,while the relative expression of CUC2 gene was opposite to the STM gene,that is the relative expression in Lenox with raised growth cone was lower than that in Longyou 7 with rscessed growth cone.3.STM and CUC2 genes are specifically expressed in growth cone and participate in the response to low temperature stress.STM and CUC2 genes were up-regulated in the stems,roots,and growth cones of Lenox and Longyou 7 compared to leaves,and the relative expression levels were highest in growth cones.The expression of STM gene in Lenox with raised growth cone was higher than that of Longyou 7with recessed growth cone,while the expression of CUC2 gene in Lenox with rasied growth cone was lower than that of Longyou 7with recessed growth cone.After low temperature stress,the relative expression levels of STM and CUC2 genes were different among different tissues.They were first down-regulated in leaves and roots,and then up-regulated.Up-regulated in stems and growth cones,and reached the highest value at-4℃,of which the up-regulation of STM gene in Lenox was more obvious than that of Longyou 7,while the change trend of CUC2 gene was contray to STM gene.Promoter function analysis showed that both STM and CUC2 genes contained multiple cis-acting elements involved in light response,stress,auxin response,and meristem expression,and STM also contained cis-acting elements involved in stress response.GUS staining showed that CUC2 gene was mainly expressed at the leaf edges,stems and growth cones of plant seedlings.4.Functional analysis after transgenic showed that STM gene had no significant effect on the growth of growth cone,while CUC2 gene inhibited the growth of growth cone,and both of them were induced by low temperature.The results showed that wild-type Arabidopsis leaves are wider than STM overexpression plants at the seedling stage,and there was no significant difference between flowering and mature stages,while there was no significant difference between CUC2 overexpression plants and wild type plants at seedling stage,and CUC2 overexpression plants at flowering stage the growth and development of the plants was slow,short plants,late fruiting,and low seed setting rate.After low temperature treatment,the survival rate and physiological activities of SOD,POD and PRO of STM overexpression Arabidopsis were better than those of wild type,and reached the highest value after-4℃ treatment for 24 h.The antifreeze experiment results of CUC2 overexpression Arabidopsis showed that the cold resistance-related physiological indexes and survival rate of CUC2 overexpressing plants were better than wild type within 12 hours after treatment at-4℃ but there was no difference between wild type and overexpression plants after 12 hours.The above results showed that STM and CUC2 were highly expressed in the growth cone of Winter Brassica rapa and induced by low temperature,in which STM gene promoted its growth by promoting the cell division of the growth cone,while CUC2,as a negative regulator of plant growth,inhibited the growth of the growth cone by inhibiting cell division,and there was a negative feedback regulation between them.