The Optimization Of Regeneration And Genetic Transformation System Of Jute And UGPase, CesAl,CCoAOMT Gene Cloning And Function Identification

Jute (Corchorus capsularis L.), also called green hemp, belong to tiliaceae, jute genera annual herb bast fiber crops, is one of the most important long staple crops in the world, the yield and planting area are second only to cotton. Because high fiber production, luster, good moisture absorption performance, quickly aproll, easy degradation, the texture of fiber is soft, which is called gold fiber in business. In recent years as breeders of continuous efforts, jute fiber production has improved dramatically, but due to low content of cellulose and high lignin content in the jute, cause the jute bast fiber cell wall lignification, single fiber cell is shorter, the texture of fiber is hard, elastic smal, lower spinning count, seriously affect the jute fiber application in textile industry and economic benefits.Plant tissue culture is the important foundation of genetic engineering, in most plants, callus is direct receptor of Agrobacterium mediated genetic transformation, also the method to obtain protoplast, somatic hybridization and plantlet regeneration. At present there are a few research reports about jute tissue culture and regeneration system. Which seriously restricts the development of jute genetic engineering.The main research content is:optimized RNA extraction sisterm using different company extract kits and methods, established high-quality RNA extraction method of jute, using optimized5’end method, we isolated the UGPase, CesAl and CCoAOMT gene and analyzed its function. Research different medium and hormone influence on tissue culture regeneration. Using phloem specific promoter to directional inhibition CCoAOMT gene. Transfer the UGPase and CCoAOMT gene into jute, tudy the possibility to improve the fiber quality of jute using UGPase and CCoAOMT gens, the main results were as follows:1.1The establish of plant regeneration and genetic transformation system of juteTake cotyledon and caulicle of "jute179" as explants to study the callus induction. The best callus induce medium respectively is:MS+0.5mg/L TDZ+0.1mg/L or0.5mg/L IAA, MS+2.0mg/L6-BA+0.5mg/L IAA and MS+0.25mg/L TDZ +0.1mg/L or0.5mg/L NAA, MS+0.5mg/L6-BA+0.5mg/L NAA. redifferentiation culture medium of cotyledon callus is MS+0.5mg/L6-BA+0.5mg/L NAA, redifferentiation culture medium of caulicle callus is MS+0.5mg/L TDZ+2.0mg/L IAA. We obetain good result using0.1%phytic acid as the browning inhibitor. The best culture medium of bud induction was MS+2.0mg/L6-BA+0.25mg/L NAA+100mg/L HC, the rate induced is36.4%, the best medium of root induction adventitious bud is MS+0.5mg/L6-BA+1mg/L NAA. Section200mg/L Cef as for inhibited the growth of the Agrobacterium after transformation,20mg/L hygromycin as screening concentration of cotyledon. At last we use fresh explant, adjusted concentration of Agrobacterium to OD600=O.5and add100μM/L AS before30min, infection time is10min, after2days, the rate of GUS staining is higher, all GUS staining plants could amplified fragment after PCR reaction. The results provides an important way for genetic engineering breeding of jute.1.2The clone and functional identification of UDP-glucose pyrophosphorylase geneIn this study we successfully cloned the full-length cDNA of the jute UDP-glucose pyrophosphorylase gene, designated CcUGPasel, using homologous cloning and modified RACE techniques. RT-PCR analysis revealed tissue difference (stem>root>leaf) and temporal differences in CcUGPasel expression, with the highest expression levels at40d and120d. Overexpression of CcUGPasel revealed increased height and more rapid growth rate in transgenic Arabidopsis compared with control lines. Importantly, the cellulose content of transgenic Arabidopsis was increased compared with control lines, although the lignin content remained unchanged. Our results indicate that jute UGPase participates in cellulose biosynthesis. These data provide an important basis for the application of the UGP gene in the improvement of jute fiber quality.1.3The clone and functional identification of Cellulose synthase(Ces41) geneWe took stem bark of jute cultivar179(Corchorus capsularis L.) as materials, successfully cloned the full-length cDNA of jute cellulose synthetase gene except500bp of5’ terminal, using homologous cloning and modified RACE techniques. The sequence length is2529bp, encoding a627amino acids protein. Gene alignment and protein structure analysis showed that it belongs to jute cellulose Synthetase gene family. Semi-quantitative RT-PCR analysis indicated that the expression level of CcCesAl gene in different parts of plant was bark> root> leaf> bud> sticks. Using partial cDNA and3’UTR region of CcCesA1gene, constructed the antisense vector of the jute CcCesAl gene, the positive plasmids were transformed into the model plant Arabidopsis thaliana. Southern blot analysis showed that the exogenous genes were transformed into Arabidopsis genome as one copy. The growth of tansgenic Arabidopsis was badly inhibited so that plants became dwarf with easing bending stem, shorter silique, and less silique numbers. This finding shows that CcCesAl gene is involved in not only cellulose synthesis, but also other plant growth process.1.4The clone and functional identification of CCoAOMT geneThe Caffeoyl-CoA3-O-methyltransferase(CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA3-O-methyltransferase(CCoAOMT) gene from jute using homology clone (Primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named "CcCCoAOMT1" Bioinformatic analyses showed the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene’s function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1in these transgenic Arabidopsis thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44-21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1gene inhibition.1.5The research on jute transformation using UDP-glucose pyrophosphorylase geneUsing Agrobacterium mediated transformation of cotyledon of jute, extract DNA from leaves of all transformed plants, after PCR and Southern hybridization detection, select7strains positive plants, we analyzed transgenic plants results showed that compared with the control lines, the transgenic plants exhibt plant higher and more content of phloem fiber cellulose than control. The results provides a new perspective to improve fiber quality of jute with UGPase gene.1.6The RNAi and miRNA silence vector construction of CCoAOMT gene and transformationThe expression result of phloem specific promoter in the tobacco show that compared with35S, phloem specific promoter can drive GUS gene expression in the phloem, and do not express or the level expression is less. Using TCK303vector and PNW55as template plasmid constructed CCoAOMT gene RNA interference vectors and artificial miRNA directional silence vector. Transformated cotyledon of jute using verified vector, at present the result of transformation are detecting, we expected to seclect transgenic plants, provide important materials for next study.