Cloning And The Expression Analysis Of Genes Related Of Rutin Biosynthesis In Juglans Sigillata Dode.

Walnut belongs to family Juglandaceae,and Juglans regia and J.sigillata are two species which were cultivated mainly.Walnuts quality is excellent and it is one of four biggest nuts(including hazelnut,almond,and cashew)in the world.As a nutrition intensive health food,walnut is rich in polyunsaturated fatty acids,proteins and secondary metabolites,such as polyphenols,alkaloids,vitamins,and so on,and has a series of pharmacological functions,such as antioxidation,antiseptic etc.Previous researches have shown that rutin was the highest content of monomers in the seed coat and leaves of walnut.There was a significant positive correlation between total phenolic content,total flavonoid content and antioxidant capacity of walnut.And the content of rutin had an important effect on total phenolic content in walnut.The synthesis and regulation of functional components of walnut using molecular biology methods has become a hot research field.In this experiment,Walnut(J.sigillata)were used as the material,Ten genes related to rutin synthesis were cloned using the leaves of the ‘Qianhe 7’ and total phenol,total flavonoids and rutin contents were measured from different tissues,including leaf,fruits,male inflorescence,female flower,root and stem,and from different developmental stages of leaf and fruits.The relationship anong enzymes activity,gene expression level and total phenolics,total flavonoids and rutin content were analyzed in order to explore the molecular mechanism of rutin synthesis in iron walnut,the results obtained in this study were as follows:(1)Ten genes,including PAL,C4 H,4CL,CHS,CHI,F3 H,F3’H,FLS,UGT and GT in the rutin biosynthesis pathway were obtained using cDNA from ‘Qianhe 7’ leaf in expansion period of as template for PCR amplification.Using the Blastp tool of NCBI for comparison and analysis,these ten structural genes have high homology with other plants.Bioinformatics,including protein molecular weight,theoretical isoelectric point,instability index,signal peptide,phosphorylation site and glycosylation site,transmembrane domain,two level structure and conservative domain,of ten structural genes were analyzed using bioinformatics analysis software.Ten genes related to rutin synthesis in iron walnut were submitted to the NCBI gene bank.The GenBank login number was respectively obtained: PAL(MG727558),C4H(MG727559),4CL(MG736731),CHS(MG736732),CHI(MG736733),F3H(MG736734),F3’H(MG736735),FLS(MG736736),UGT(MG736737),GT(MG736738).(2)GAPDH,β-Actin,18 S rRNA,UBQ and EF-1 which were commonly used in plant as reference genes,were expressed and analyzed in Walnut(Juglans spp.).GAPDH and EF-1 showed stabliliy in all samples of walnut,and were suitable for target gene expression with low abundance,due to their low abundance of expression.On the contrary,18 S rRNA was suitable for the gene expression with high abundance.β-Actin and UBQ were not suitable for analysising gene expression in walnut for their poor stability.(3)The contents of total phenol and rutin in the two varieties’ s testa were significanty higher than those in other organs,while the content in cotyledon was the lowest.The total flavonoids in leaves was significantly higher than that of other organs,seedcases was the lowest.The contents of total phenols in leaves of two cultivars both showed an upward trend,after anthesis 88 ~ 119 d,‘Qianhe 7’ showed slightly doenward trend.Rutin fluctuates greatly,and the change rule was not obviousily.The variation tendency of phenol and rutin contents in greenhouse was descends firstly,then rises,then drops at last.The total phenol content in testa increased first,then slightly decreased and increased finally.While the content of rutin always rised.In cotyledons the content of total phenols rised firstly,slightly fell.The content of rutin decreased firstly,then increased,and decreased finally.(4)In the study of spatial expression of J.sigillata,it was found that the activities of ten related enzymes are all tissue specific,The activities of C4 H,4CL,CHS,CHI,F3 H,UGT and GT were similar in two varieties,and their activities were highest in stems.The expression of ten enzyme genes was also tissue-specific,The expression of CHI,F3 H were the highest in the female flowers,F3’H,UGT,GT in pellicle,C4 H in root were the highest,respectively.While the expression level of PAL in leaves and CHS in stems were the lowest.The difference of gene expression among the two varieties was mainly reflected in PAL,4CL,CHS and FLS.The genes of PAL,4CL and FLS were the highest in male flowers,and CHS was highest in roots of ‘Qianhe 7’.While the expression of PAL in stems,4CL in roots,CHS and FLS in leaves were highest.(5)The content of polyphenols was negatively correlated with the activity of related enzymes(except UGT)during the development of the leaves development,and the correlation was significantly different from that of PAL,4CL,CHS,CHI,F3 H,F3’H,and FLS enzyme.The expression of PAL、CHI、F3H、F3’H、UGT were significantly negatively correlated with the content of polyphenols.While the expression of FLS,GT were a significant positive correlation with the content of polyphenols.During the development of green husk,he content of polyphenols was positively correlated with the activities of most related enzymes,PAL、C4H、4CL、CHI、FLS、UGT、GT,especially.The expression of PAL、F3H、FLS were significantly negatively correlated with the content of polyphenols in green husk.During the development of pellicle,The content of polyphenols was positively correlated with C4 H,CHI,F3 ’H,FLS,UGT and GT enzyme activities,and negatively correlated with 4CL enzyme activity.While the expression level of CHS,FLS and GT was positively correlated with the content of polyphenols.During the development of kernel,the content of polyphenols was very low,and the correlation with relative enzyme activity was weak.There was only a significant positive correlation with the genes expression of CHS.