The Mechanism Of Cold Resistance Of Winter Rape(Brassica Napus L.) Based On Growth Point And Hypocotyl

Brassica napus L.is one of the main oil crops in China,which has the characteristics of high yield,lodging resistance and good quality.During the seedling period before overwintering,the hypocotyls elongated,resulting in a large volume of growth point exposed to the soil surface,and the plant was easily killed by freezing,which seriously restricted the development of Brassica napus in northern China.Therefore,the study of cold resistance of Brassica napus based on growth point and hypocotyl is of great significance to analyze the molecular mechanism of cold resistance of Brassica napus.In this study,12 winter rapeseed?Brassica napus?were used as research Material,and their cold resistance was identified by overwintering rate,vernalization rate,semi-lethal temperature,morphological characteristics,physiological and biochemical indexes,etc.and the correlation between the relative height of growth point&the length of hypocotyls and cold resistance was identified,and the functions of the selected key candidate genes regulating growth point differentiation and hypocotyl elongation were analyzed.The main results are as follows:1.The results of overwintering test and vernalization test showed that there was a very significant negative correlation between overwintering rate and vernalization rate of Brassica napus?the correlation coefficient was-0.964?,in which the overwintering rate was 7.60⁹2.15%,and the vernalization rate was 11.65⁹7.14%.Under low temperature treatment,the relative conductivity of the tested Material increased,the semi-lethal temperature(LT₅₀)was-12.84?^-2.57?,the enzyme activity and the content of cellular osmotic regulators increased significantly,the content of chlorophyll decreased and the content of carotenoids increased.Hierarchical cluster analysis was used to analyze the cold resistance of 12 Brassica napus,and the results showed that the cold resistance of 12 Brassica napus could be divided into four categories:strong cold resistance materials?TSG 10,TS309-4,NS45-4,TS309-3,TS309-10and NTS15?,cold resistance materials?8?G?,TS312-2 and Tianyou 2266?,weak cold resistance materials?NPZ269-1 and Tianyou 2288?and non cold resistance material?Xinyou 23?.2.There are great differences between growth point and hypocotyl of Brassica napus.Strong cold-resistant material?such as TSG 10?have low growth point height,smooth and transparent surface,hemispherical steamed bread shape,slow differentiation ability,small volume,compact growth point cells,dense cytoplasm and large nuclei,while weak cold-resistant material?such as Tianyou 2288?have larger growth point height,many young tissue protuberances on the surface,strong differentiation ability and large volume,and the cells at the base of the growth point are loosely arranged and dispersed and the cell gap is large.Hypocotyl cells at extremely low temperature?-20??,there are a large number of cavities and collapsed cells in the cell cortex,and the cell wall is fused and severely damaged.3.The relative height of growth point and hypocotyl length of Brassica napus were greatly affected by leaf age,low temperature treatment and sowing date.With the increase of leaf age,the relative height of growth point increased,the lignification range of hypocotyl near root gradually extended to shoot,and the length of hypocotyl became shorter in low temperature treatment?4??than in normal temperature?25?20??.The relative height of growth point and the elongation rate of hypocotyls in autumn sowing treatment were smaller than those in spring sowing treatment,and the hypocotyl length was shorter in low temperature treatment?4??than in normal temperature?25?20??.Correlation analysis showed that the relative height of growth point and the length of hypocotyl were significantly negatively correlated with cold resistance.4.Low temperature treatment?4??than in normal temperature?25?20??,the key candidate gene for regulating growth point differentiation is BnSTM gene,and the key candidate gene for regulating hypocotyl length is BnHY5 gene.The expression of BnSTM gene was positively correlated with the growth point and negatively correlated with the cold resistance of Brassica napus.BnHY5 gene was mainly expressed in leaves and hypocotyls.5.The regulation of growth point differentiation by proteins encoded by BnSTM gene is mainly accomplished by the concentrated expression of KNOX family in stem meristems,while the regulation of hypocotyls length by proteins encoded by BnHY5 gene is closely related to BRLZ transcription factors of bZIP superfamily.The CDs sequences of BnSTM,a positive regulatory gene for growth point differentiation,and BnHY5,a positive regulatory gene for Hypocotyl elongation,were cloned.The results of protein structure and function prediction showed that BnSTM gene contained 1149bp complete ORF,encoding 382 amino acids,belonging to the KNOX family,including four conserved domains:KNOX1,KNOX2,ELK and Homebox KN,and was closely related to Tianyou 2288,Brassica napus,Brassica oleracea L.and Brassica rapa L.BnHY5 gene contains 528bp complete ORF,protein encoding 175amino acids,which belongs to the bZIP superfamily and contains a conserved domain of BRLZ transcription factors,which is closely related to Tianyou 2288 and Brassica napus.Subcellular localization analysis showed that both BnSTM and BnHY5 proteins were mainly expressed in the nucleus,which was consistent with the predicted results.At the same time,T1 generations of transgenic BnSTM and BnHY5 Arabidopsis positive lines were successfully identified.These results showed that the relative height of growth point and the length of hypocotyls were closely related to the cold resistance of Brassica napus,and the key candidate genes BnSTM of growth point and BnHY5 of hypocotyls were successfully identified,which provided a new direction for cold resistance breeding of winter rape materials of Brassica napus.However,the molecular mechanism of BnSTM and BnHY5 genes in cold resistance needs to be further studied.