| Brassica genus of Cruciferae family includes many important vegetable crops and oil crops,of which Chinese cabbage(Brassica rapa L.)is a representative vegetable crop and B.napus is a representative oil crop.With the continuous deterioration of the global climate in recent years,abiotic stresses,such as high temperature and drought,seriously affect the yield and quality of Brassica crops.The development of varieties with heat-and drought-resistance is the main way to alleviate the threat of abiotic stress.In the model plant Arabidopsis thaliana,micro RNA398a(mi R398a)regulates plant heat tolerance by silencing target gene CSD(COPPER/ZINC SUPEROXIDE DISMUTASE)family,and JAZ5(JASMONATE ZIM-DOMAIN 5)genes are related to plant drought resistance.However,do the mi R398/CSD family genes of Chinese cabbage and the JAZ family genes of rapeseed participate in heat drought-resistance,respectively? Do members of the CSD family or JAZ family have the same functions? Can they be used in heat resistance breeding in Chinese cabbage and drought resistance breeding in rapeseed? To answer these questions,in the present study,we identified the homologous gene Bra-MIR398 a of At MIR398 a in Chinese cabbage and the homologous gene Bn A.JAZ5 of At JAZ5 in rapeseed,studied the functions of mi R398/CSD or JAZ5 genes in Brassica,to clarify the functions of these two genes in heatand drought-resistance traits.The findings will provide scientific basis and genetic resources for heat-resistance breeding in Chinese cabbage and drought-resistance breeding in rapeseed.The main results obtained are as follows:1.There are three MIR398 loci in Arabidopsis genome,namely MIR398 a,MIR398b and MIR398c;There are three mi R398 target genes,namely CSD1,CSD2 and CCS.We identified four MIR398 genes in Chinese cabbage,predicted their six target gene loci in Chinese cabbage,and named them as Bra CSD1-1,Bra CSD1-2,Bra CSD2-1,Bra CSD2-2,Bra CCS-1and Bra CCS-2,respectively.Moreover,the degradome data analysis,5’ RACE and q RT-PCR experiments proved that Bra CSD1-1 was the target gene of Bra-mi R398 a.2.Chinese cabbage mi R398 a and target gene CSD1 were cloned.Binary transgenic vectors with over expression of Bra-mi R398a(p35S::a Bra-mi R398a)and Bra CSD1-1(p35S::Bra CSD1-1)and low expression of Bra-mi R398a(p35S::Bra-MIM398a)were constructed,respectively.The corresponding transgenic lines were obtained by transforming Chinese cabbage(Bre)by vacuum filtration.Under heat stress,overexpression of Bra-mi R398 a transgenic lines delayed the time of leaf death and increased the survival rate of Chinese cabbage under stress,while low expression of Bra-mi R398 a and overexpression of Bra CSD1-1 accelerated leaf death and reduced the survival rate.3.q RT-PCR results showed that the expression of heat stress-related genes Bra LEA76,Bra Ca M1,Bra PLC,Bra DREB2 A,and Bra P5 CS were up-regulated in the p35S::a Bra-mi R398 a transgenic lines.However,these genes were down-regulated in the p35S::Bra-MIM398 a transgenic lines.This result indicated that Bra-mi R398 a improving the heat resistance of Chinese cabbage by up-regulating the expression of heat stress-related genes.4.Two genes Bn A.JAZ5 and Bn C.JAZ5 in B.napus,homologous of At JAZ5 in Arabidopsis,were found.Under drought stress,the expression of Bn A.JAZ5 was significantly enhanced,while the expression of Bn C.JAZ5 gene did not.This result indicated that Bn A.JAZ5 is the main functional allele.5.Bn A.JAZ5 of B.napus was cloned,and its overexpression transgenic lines(p35S::Bn A.JAZ5)were obtained by hypocotyl transformation.Under drought conditions,the death rate of Bn A.JAZ5 overexpression lines was increased.Using 400 m M mannitol to simulate drought stress,the germination rate,cotyledon opening ratio and root elongation of p35S::Bn A.JAZ5 were lower than those of wild-type.Under drought stress,the expression of drought stress-related genes Bn P5 CS,Bn PLC,Bn CYSTM1,and Bn LEA76 in p35S::Bn A.JAZ5 transgenic rapeseed seedlings was lower than that of wild-type.Moreover,the stomatal density on the leaf surface of transgenic rapeseed lines was higher than that of wild-type,and the stomatal closure ratio under drought stress was lower than that of wild-type.In addition,the water loss rate of transgenic rapeseed lines was higher than that of wild-type,and the relative water content under drought stress was lower than that of wild-type.These results indicate that Bn A.JAZ5 gene regulates the drought resistance of rapeseed by affecting the expression of drought stress related genes and the stomatal response on the leaf surface.6.The expression of Bn A.JAZ5 gene was significantly up-regulated under jasmonic acid(JA)treatment.However,the expression of Bn A.JAZ5 gene did not change significantly under abscisic acid(ABA)treatment.Under drought stress,overexpression of Bn A.JAZ5 significantly changed the expression level of the JA pathway specific response gene Bn MYC2,but did not significantly change the expression level of the ABA pathway specific response gene Bn ABF3.This result indicated that Bn A.JAZ5 was involved in JA signal transduction pathway and regulated drought resistance of rapeseed in an ABA dependent manner.This study found that the Bra-mi R398a/Bra CSD1-1 module plays an important role in the heat resistance of Chinese cabbage.Under heat stress,the over expression of Bra-mi R398 a increases the heat resistance of Chinese cabbage by prolonging the leaf death time,while the over expression of Bra CSD1-1 shortened the leaf death time and weakened the heat resistance of Chinese cabbage.Overexpression of Bn A.JAZ5 reduced the drought resistance of rapeseed through ABA-dependent pathway.The findings will enrich the theory of stress resistance of Brassica crops,and provide a theoretical basis and candidate genes for developing high-quality,high-yield and stress-tolerant Brassica crops. |
