| Jatropha curcas(Jatropha curcas L.),a species of Euphorbiac eae,has great potential in refining biodiesel for its high seed oil content(60%).However,the low ratio of female to male flowers of J.curcas(about 1:29)is thought to be responsible for its low fruit set.Plant hormones play important roles in plant growth,differentiation and reproductive development,as well as a key role in the process of flower sex differentiation.Brassinolide is a kind of promoting hormone,which plays an important role in agricultural production,can improve plant resistance to disease,salt and frost resistance.BRI1 is a receptor protein of BRs located on the cell membrane,and BRL1 and BRL3 also have the function of recognizing and combining BRs.It is worth noting that BAK1,a common receptor of BRI1,is involved in the regulation of a variety of LRR kinases,and the interaction between different signaling pathways can be achieved through the role of BAK1 protein.In the previous work,a transcriptome from flower buds of J.curcas at different developmental stages was constructed,and a homologue of BRL3 gene was screened out from the transcriptome.In this study,the BRL3 gene of J.curcas was cloned on the basis of the selected unigene fragments.The sequence and spatio-temporal expression patterns of the J.curcas were analyzed,and the preliminary functional validation of the plant overexpression vector was constructed to the tobacco body.The main results were as follows:1.The full-length cDNA sequence of BRL3 gene was obtaining from flower buds of J.curcas,using RACE technology,and named as JcBRI1-like3(JcBRL3)which has a total length of 4755 bp.The open reading frame of this gene is 3618 bp long,and is estimated to encode a protein with 1205 amino acids;this protein was predicted to has a theoretical molecular weight about a mass of 130.71 kDa,and a theoretical isoelectric point of 6.06.2.The pET-30a/BRI1-like3 prokaryotic expression vector was successfully constructed,and the recombinant JcBRL3 was expressed.The results showed that the JcBRL3 gene could be expressed in the E.coli BL21 and E.coli Rosetta under different induction conditions.The induced protein was identified using LC-MS/MS.The results further suggested that this protein was BRL3 protein.The analysis of amino acid sequences showed that JcBRL3 protein is a leucine rich repeat receptor kinase protein.This protein was located at membrane under the guidance of the N terminal signal peptide sequence of it;there is a BRs binding site at the extracellular fragment,and a functional domain at the trans-membrane motif of it.Amino acid multiple sequence alignment and structure prediction analysis showed that JcBRL3 protein was highly homologous with BRI1 protein.Therefore,JcBRL3 gene was a BRL3 protein gene.3.qRT-PCR was used to analyze the expression profiles of JcBRL3 at different development stages of flower(such as the period of female flower megaspore mother cell,the period of female flower macrospore mother cell division,the mononuclear embryo sac period,the female flower embryo sac maturity stage,the male flower microspore mother cell period,the male flower four division period,the male flower mononuclear pollen period and the male flower)with the undifferentiated period of the flower bud of J curcas as the control group.JcBRL3 gene showed a “rising and descending” trend in the female flower,peaking at the mononuclear embryo sac period.In the male flower,the expression level of the JcBRL3 gene was characterized by a “rising – descending” trend,peaking in the mature period of pollen grains with a higher expression level than that of other development periods of flower.4.The plant overexpression vector pBWA(V)KS-BRI1/like3 driven by CaMV35 S promoter was constructed,and transformated to the tobacco K326.The morphological structure analysis of transgenic tobacco showed that the length of the stamen was significantly longer than that of the wild type tobacco,suggesting that the excess expression of the JcBRL3 gene could promote the development of the stamen. |
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