Cloning And Preliminary Function Analysis Of The Auxin Efflux Carrier CcPIN Family Genes In Chinese Hickory(Carya Cathayensis Sarg.)

Chinese hickory(Carya cathayensis Sarg.),as a woody oil tree species,belongs to the genus Carya Nutt.in Juglandaceae.Chinese hickory has high economic value and its industry is one of the economic ways to get rid of poverty for farmers in the mountainous areas of Northwest Zhejiang.However,problems such as tall tree,long juvenile period and low improvement level has restricted the development of Chinese hickory industry seriously.The application of grafting technique can solve the above problems effectively.Previous studies have revealed that auxin plays an important role in the process of graft healing,but how polar auxin transport mediated by auxin transport carriers participates in the graft healing process is little reported.As auxin efflux carriers,PIN proteins are mainly involved in the intracellular and intercellular transport of auxin.Cloning Cc PIN genes from Chinese hickory and exploring their preliminary functions can lay a foundation for revealing the regulatory mechanism of Cc PINs during the healing process of Chinese hickory grafting.In this study,Cc PIN family genes were cloned based on the previous sequencing data and their sequence characteristics were analyzed;the expression patterns of Cc PIN family genes during the grafting process of Chinese hickory were identified;the cis-acting elements on the promoters of Cc PIN family genes as well as the inducible activities of Cc PIN3 gene promoter were analyzed;the functions of Cc PIN3 gene was preliminarily explored by heterologous transformation of Arabidopsis thaliana.The main results are as follows:(1)The full-length CDS sequences of six Cc PIN genes were cloned.4-5 transmembrane helical structures were detected at the C-terminal and N-terminal of Cc PIN family proteins,respectively.Cc PIN proteins were more closely related to the homologous proteins from poplar.(2)The expression trends of Cc PIN genes in rootstocks and scions were inconsistent during the grafting process of Chinese hickory.In scions,the expressions of most Cc PINs showed a significant change in the whole grafting process.The expression levels of Cc PIN3,Cc PIN4 and Cc PIN6 were lower in the early stage(3 days)of grafting,and significantly higher in the late stage(14 days),which were 8.0 times,5.3 times and 3.3 times of those before grafting.In rootstocks,the relative expression levels of Cc PIN3,Cc PIN6 and Cc PIN8 genes changed significantly during the whole grafting process,among which the relative expression levels of Cc PIN3 were the highest on the 7th day after grafting,which was 3.5 times of that before grafting,showing an overall expression trend of first increasing and then decreasing.During the whole healing process,the expressions of Cc PINs were higher in scions than that in rootstocks.Under the treatment of exogenous hormones IAA and NPA,the expression patterns of Cc PIN family genes in scions and rootstocks were changed.In general,the expression levels of Cc PINs in scions were higher than those in rootstocks.Tissue-specific expression analysis of Cc PINs showed that the expression levels of Cc PIN family genes were relatively high in root and leaf,but low in stem.(3)Three main types of cis-acting elements on the promoters of Cc PIN family genes were detected,which were hormone response related auxin,gibberellin,methyl jasmonate,salicylic acid,zeatin and abscisic acid elements;stress response related light response and drought response elements;and development related meristem expression,flavonoid biosynthesis,physiological control,and endosperm specific expression elements.The inducible activities of Cc PIN3 promoter were related to its length.Cis-acting elements promoting the inducible activity were detected in the promoter region of-2 000 to-500bp;while no expression activity were detected in the region of-500 to-1bp,indicating that cis-acting elements were insufficient to driven the inductible activity in this region.The expression of GUS gene was detected in the root of A.thaliana with the transient transformation of Cc PIN3 promoter,which verified the activity of Cc PIN3 promoter in regulating gene expression.(4)Three A.thaliana lines overexpressing Cc PIN3 were successfully obtained.Morphologically,the root length of the overexpressing lines was about 1.4 times that of the wild type;the leaf length,leaf width and petiole length of the overexpressing lines were 1.3,1.4 and 1.2 times that of the wild type,respectively;the height of transgenic lines at 45 d and 60 d was 72.5% and 77.5% that of the wild type,respectively;the cross-sectional area of the stems of the three overexpressing lines was 3.2,3.5 and 2.3 times that of the wild type,respectively.Further stem sections showed an increase in overall cell size,cell number,and cell wall thickness in the transgenic lines.These phenotypic changes in the transgenic lines were associated with multiple pathways such as plant hormone signal transduction,flavonoid biosynthesis and ABC transport pathways.