| The early maturing rapeseed in the double-cropping rice area is an important rapeseed production basement in china.Low temperature(chilling and frost)is an important environmental factor to limit yield of rapeseed in this area,causing slow growth of rapeseed in autumn and winter.It is vulnerable to early spring cold current and sudden temperature drop in autumn bring huge loss.To date,there are many researches on the genetic and molecular mechanisms related to low-temperature tolerance in rapeseed.The study of the genetic law of low-temperature-tolerant germplasm in rapeseed is aimed at providing reference for the genetic improvement of low-temperature tolerance rapeseed varieties.In this study,we screened low-temperature-tolerant germplasms in response to low-temperature and chilling damage in the Yangtze River Basin,and explored the inheritance of low-temperature and chilling-tolerant germplasm through the study of germplasm under low temperature stress.Previous studies have shown that lightharvesting chlorophyll a/b-binding proteins(Lhcb)not only function in plant photosynthesis,but also regulate the plants response and resistance to abiotic stress.In order to investigate whether Lhcb in rapeseed responds to low temperature stress,the transcriptome data of the excellent low-temperature resistant germplasm C18 and the sensitive variety ZS6 were used to screen the Lhcb genes that responded to low temperature stress,and the Lhcb family genes were analyzed.Therefore,in order to solve the problem of low temperature tolerance and explore the function of Lhcb in response to low temperature stress,the main research results are as follows:(1)In order to improve low temperature tolerance of rapeseed,10 rapeseed inbred lines with different level of low temperature tolerance were selected,and 25 combinations were generated by NCⅡincomplete bi-serial hybridization.Seeds of 10 parents and 25 combinations were germinated under normal condition(22°C)and low temperature condition(9°C),germination rate,germination potential,germination index,mean germination were investigated to analyze combining ability,genetic parameters and heterosis of various germination traits.Results showed that there were significant differences in germination traits among different hybrid combinations at 9℃,but no significant differences were detected at 22℃.P2(Ning You 18),P7(2007R13)and P10(C18)had strong germination capacity at 9℃,and higher general combining ability(GCA)of each trait.The hybrid combination generated by P2(Ning You 18),P7(2007R13)and P10(C18)had higher special combining ability(SCA)of each germination trait and strong heterosis at 9℃.GCA of the four traits in cultivar C18 was the highest,and cross(B018×Huyou 17)×C18 exhibited the highest of special combining ability(SCA).Analysis of genetic parameters showed that heritability of the relative germination index in the narrow sense was larger than that in the broad sense,and should be selected in the early generation.The narrow sense heritability of relative germination potential,relative germination rate and relative average germination time is lower,while the broad sense heritability is relatively high,so it should be selected in the late breeding generation.The broad heritability and narrow heritability of germination index were the highest among the four traits.Overall,combining ability and genetic effects analysis of germination index during germination stage can be used to identify low temperature tolerance germplasm of rapeseed to improve the breeding program.In addition,through the comprehensive comparative analysis of the low temperature tolerance of the selected parents,it was found that P10(C18)had the strongest resistance to low temperature,and P3(ZS6)was the most sensitive to low temperature.(2)By using transcriptome data from C18 and Zhongshuang 6,we identified 35 light-harvesting chlorophyll a/b-binding proteins(Lhcb)family members in rapeseed.The evolutionary analysis of the sequence characteristics,physicochemical properties and chromosome positions of these Lhcbs proteins were conducted in rapeseed and Arabidopsis.There were 35 Lhcbs genes in rapseed with the length of234 aa～329 aa,which could be divided into 8 subfamilies.The physicochemical properties of the BnLhcb proteins in the same subgroup were similar.35 BnLhcbs protein genes distributed on 15 chromosomes.No Lhcb gene was found on C01,C06,C10,A01 and A04 chromosomes,BnLhcbs genes were distributed on 8 chromosome of subgenome A and 7 subgenome C,with each chromosome containing 1 to 4 BnLhcbs genes.By comparing the expression levels of 35 BnLhcbs in a sensitive variety ZS6 and a cold resistance variety C18 transcriptome data under low temperature condition,we found that the expression level of the nine genes(BnLhcb2.1、BnLhcb2.2、BnLhcb2.3、BnLhcb2.5、BnLhcb2.6、BnLhcb2.7、BnLhcb3.4、BnLhcb6.1 and BnLhcb6.2)in C18 was significantly higher than that of ZS6.Among them,BnLhcb3.4had the most significant difference between the two materials and played an important role in the response of rapeseed to low temperature stress.Therefore,BnLhcb3.4 gene was selected for futher transgenic study.(3)In order to verify the function of BnLhcb3.4 in low temperature tolerance,the subcellular localization analysis of BnLhcb3.4,gene showed that the gene was localized in chloroplast.The BnLhcb3.4 overexpression vector was constructed and successfully transformed into Arabidopsis.The homozygous line,lhcb3 mutants and wild-type plants were treated with at-5℃ for 2 h,and the growth was resumed at room temperature for 5 days.The survival rate,photosynthetic parameters,physiological and biochemical indexes and the expression levels of genes related to low temperature response were analyzed.The results showed that the survival rate of the BnLhcb3.4 overexpression plants was significantly higher than that of wild-type plants and lhcb3 mutants after low-temperature stress.The further study indicated that BnLhcb3.4 improved the low temperature tolerance of Arabidopsis by increasing the content of osmoregulatory substances(soluble sugar,proline)and decreasing the content of membrane lipid peroxidation products(electrolyte permeability,malondialdehyde).Moreover,BnLhcb3.4 significantly increased the expression of genes related to low temperature response,including ABA signal transduction pathway key genes,CBF signal transduction pathway key genes.Taken together,the results indicated that BnLhcb3.4 played an important role in the response to low temperature stress in plants,which provided gene resources for breeding low temperature tolerance of rapeseed. |
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