Expression Analysis Of Secondary Cell Wall Synthesis Related Genes And Functional Identification Of CcCOBL1 In Jute

Jute(Corchorus capsularis)(2n=14),which is one of the important fiber crops in the world,belongs to Corchorus genus in the family of Malvaceae.The content of jute cellulose determines its fiber quality.In this study,an elite cultivar,Huangma 179,was used as materials,and the realtime fluorescence quantitative internal reference genes of different parts of jute in different periods were screened based on the self-assembled genome data of the research group.Then,the secondary cell wall genes of jute were identified and their expression patterns were analyzed.At the same time,the CcCOBRA gene family of jute was also identified,and the expression patterns of CcCOBRA gene family in different stem development were analyzed.The function of PSN domain in the CcCOBL1 gene of jute was verified,and the jute gemplasme were obtained by transferring the PSN domain of CcCOBL1 gene.The results are as follows:1.In order to screen the internal reference genes of different parts of jute in different periods,eight candidate internal reference genes were preliminarily screened by Arabidopsis homologous genes and combined with the existing Huangma 179 transcriptome data of the research group.Through the coefficient of variation of FPKM value of different candidate internal reference genes in different growth periods,the stability ranking of these internal reference genes is as follows: TUBα(9.3%)< ACT(6.6%)< Dna J(6.2%)< TUBβ(6.1%)< UBQ(4.9%)< EF1α(4.2%)<UBC(3.7%)< UBI(2.8%).In order to further verify the data of electronic expression,these genes in the roots,stems and leaves of Huangma 179 tissues at the stage of 14 days after germination were detected by fluorescence quantitative PCR(qRT-PCR).The stability of 8 candidate genes was verified by the coefficient of variation of Ct value,the software of ge Norm and the software of Norm Finder.The results showed that the stability of the internal reference genes screened by transcriptome was basically consistent with the stability of the three methods,and the method was feasible to a certain extent.The best combination of internal genes was obtained: Dna J + UBQ + UBI.2.Moreover,8 secondary cell wall synthesis genes were identified,including lignin synthesis genes: Cc4CL1 and CcCCo AOMT1;cellulose synthetase genes: CcCesA4,CcCesA7,Cces A8;xylan synthesis genes:xylan synthesis genes: CcCcFRA8,CcIRX8,CcIRX9.The electronic expression of these genes in hypocotyls on 10 days,stem barks on 60 days and stem barks on 90 days after germination were analyzed.It was found that the expression of lignin synthesis gene began to decrease slowly after60 days after germination,while the expression of cellulose synthetase genes and xylan synthesis genes expect CcFRA8,decreased at 60 days after germination,and decreased sharply at 90 days.The expression patterns of lignin synthesis gene and cellulose synthetase gene in stems at different stages(7d,14 d after germination)were analyzed by qRT-PCR.It was found that 5 genes had been expressed at seedling stage,and the expression of 5 genes on the 14 th day was higher than that on the 7th day after germination.It also confirmed the practicability of the internal reference gene.3.Expression changes of COBRA genes could affect the content of cellulose and cell oriented growth in plants.It is imperative to identify CcCOBRA gene in jute.Totally,15 CcCOBRA genes of jute were identified,named CcCOBL1 ～ CcCOBL15.The results showed that the CcCOBRA family was divided into two subfamilies.All the CcCOBRA family members have specific domain(PF04833),including 7 family members with N-signal peptide and 4 family members with GPI anchor.15 COBRA genes have different expression levels in hypocotyls of 10 days,stem bark of 60 days and stem bark of 90 days after germination.Particarly,the expression levels of CcCOBL1,CcCOBL12 and CcCOBL14 increased gradually.The expression of CcCOBL1 in jute seedlings was analyzed by qRT-PCR.It was found that the expression of CcCOBL1 in jute seedlings had begun,and the expression level at 14 days was higher than that at 7days.4.In order to verify the function of CcCOBL1 with the increasing expression of stems in growth stages,we constructed the overexpression vector containing PSN sequences of CcCOBL1 gene,and then transferred the target into Arabidopsis by Agrobacterium,and got 2 Arabidopsis lines with the target gene.40 days after germination,the plant height and the cellulose content in leaves of transgenic Arabidopsis lines were higher than that of the control.The results suggested that the PSN domain of CcCOBL1 gene was involved in the development of plant fiber.At the same time,the transgenic jute germplasm with PSN domain of CcCOBL1 gene was also obtained by Agrobacterium using stem tip.The above results will provide candidate genes for further researches on fiber development and facilitate breeding-by-design of fiber quality improvement in jute.