Cloning And Tissue Expression Analysis Of GalAT And UGlcAE Genes In Ramie (Boehmeria Ivea (Linn.) Gaud.)

Ramie [Boehmeria nivea(Linn.)Gaud.] bast fiber is a kind of fine plant textile materials with chinese feature,however, staple chemical degumming means in ramie for industrial production have many such shortcoming as lower bast fibre strength, lower essential fiber productivity, deterioraten spinnability of fibre; prolong process flow; heighten deguming costs ; increase alkali content of boiling-epurate exhausted liquid, pollute surroundings severely; consume energy source heavily, all of which restrict severely development of bast fiber spin industry. Pectin is one of primary colloidal matter of ramie phloem,and its content next to hemicellulouse in colloidal substance. Pectin stick all kinds of colloidal substances together, which make ramie another colloidal substances to unfix more difficultly, so removing pectin is one of keys in ramie degumming process. Lower pectin content moderately is a important direction of ramie high quality breeding at present. GalAT(α-1,4-Galacturonosyltransferase) and UGlcAE(UDP-glucuronic acid-4-epimerase) are two key enzymes in pectin and pectin major component biosynthesis,respectively. Thus,some researches on gene sequence and tissue expression characteristic of GalAT and UGlcAE gene have practical significance to regulate and control pectin content by molecular biology method in our later experiment..In the research, SMART technology was used to construct full length cDNA library of ramie phloem; Degenerate primer RT-PCR method was used to clone cDNA core sequence of goal gene;RACE technology and screening method of full-length cDNA library were used to obtain full length cDNA sequence;Bioinformatics methods were used to analyze cDNA sequences obtained and putative amino acid sequences; Fluorescence real-time quantitative PCR method was used to research gene expression in different tissues. Main research results as follows:(1)Phloem full length cDNA library of ramie fine variety: Zhongzhu NO.1 was constructed. Titer of primitive library was 1.25×105pfu /mL,recombination rate was 92%,titer of the amplified library was 1.13×109pfu /mL. The result set up a fundament to clone full length sequence of fine gene from ramie and research its genetic rule.(2)Three cDNA fragments of GalAT gene were cloned, which sequence length were 1087bp,986bp and 238bp,respectively.The 986bp sequence was sent to Genbank on line and acquired accession number:EU131377.The sequence encoded 328 successive amino acid sequence. Analyse of conservative region by NCBI on line proven that conservative region:Glycosyltransferase family 8 was included in mRNA sequence; Blastn comparison showed that homologue between the sequence and GAUT4(one of GalAT family genes) in Arabidopsis thaliana was the highest for 83%;Multi-sequence comparison analyse showed that GalAT gene putative amino acid sequence had some conservatism between ramie and Arabidopsis thaliana;Molecular evolution analyse showed that GalAT gene putatived amino acid sequence in ramie and GAUT4 in Arabidopsis thaliana was gathered to same group.(3)Three cDNA fragments of UGlcAE gene were cloned, which sequence length were 308bp,410bp and 262bp,respectively.(4)Full length cDNA sequence of UGlcAE gene was cloned based on 410bp cDNA sequence which had been obtained in above experiment.The cDNA was composed by 1257bp sequence,and its ORF(open reading frame) which encoded 241 amino acids between 443bp and 1165bp location of mRNA sequence. GeneBank accession number was EU131378.The pI and molecular mass of putatived protein were 9.86 and 26kD,respectively.NAD(P)binding site,NAD-epimerase/dehydratase and UDP-galactose-4-epimerase domain were discovered in putatived protein by InterPro analyse; Conservative region analyse by NCBI on line proven that high conservative region: UDP-galactose-4-epimerase was discovered in coding region. BlastN analyse showed that cDNA sequence of ramie UGlcAE gene shared 84% identity with Zea mays corresponding sequence; BlastP analyse showed that homologue of amino acid sequence between ramie UGlcAE gene and GAE6(one of UGlcAE family genes) in Arabidopsis thaliana was the highest.Multi-sequence comparison analyse showed that UGlcAE gene putative amino acid sequence had high conservatism between ramie and some other species.Molecular evolution analyse showed that the putative amino acid sequence and Arabidopsis thaliana GAE 6 was gather to same group..(5)Tissue expression distribution of GalAT gene in ramie fine variety: Zhongzhu NO.1 was researched by real-time fluorescence quantitative PCR method. Result showed that GalAT gene could expressed in all kinds of ramie tissues, and its mRNA was abundant in root, leaf took second place,phloem and xylem were the least and had no significant difference between them.(6)Tissue expression distribution of UGlcAE gene in ramie fine variety:Zhongzhu NO.1 was researched by real-time fluorescence quantitative PCR method. Result showed that UGlcAE gene could expressed in all kinds of ramie tissues, and content of its mRNA in all kinds of ramie tissues in turn revealed as follow: root>leaf>phloem>xylem.(7)Tissue expression of GalAT and UGlcAE gene were compared, the result showed that tissue expression of the two gene were same approximately,for instance,content of the two gene mRNA appeared as follow:root〉leaf〉phloem〉or≈xylem,and were the most abundant in root,but mRNA number of GalAT gene was 0.6533 times more than that of UGlcAE gene in phloem.We think that molecule regulate and control test on ramie pectin in later stage should begin from GalAT gene or proceed two genes cosuppression simultaneity.In conclusion,the research has acquired some progress in cloning key enzyme genes:GalAT gene,UGlcAE gene in ramie pectin biosynthesis and their tissues expression character, which established a fundament to acquire fine ramie new variety with lower pectin content by regulate and control pectin content from molecular level in our further research.