The Cloning Of PHL And AHP5 Genes And Their Functional Analysis In Sweet Orange And Strawberry

The level of sugars,acids and the sugar/acid ratios in fruit are critical contrbutors to fruit sweetness,a major quality trait for various fruits.The major goal of this study is to characterize the functions of PHL(PH-LIKE)and AHP5,two genes known to be related to the control of fruit acidity and sugar/acid ratios.PH was first identified from a recent molecular genetic study in sweet melon as a critical regulator of fruit acidity.It encodes a protein most closely related to the Arabidopsis auxin efflux carrier family(PILS).However,the biochemical function of PH remains a mystery because PH protein has not been shown to exhibit any transporter activity and the PH silencing transgenic plants did not exhibit any phenotypes related to auxin.AHP5 was reported as a candidate gene may involved in the control of sugar/acid ratios from a prior sweet orange gene co-expression network study conducted in our laboratory.To speed up discovery pace,we performed expression and functional studies using both sweet orange(Citrus sinensis)and diploid strawberry(Fragaria vesca)given the much shorter junvenile phase in strawberry than orange.Because the protein encoded by PH gene is related to auxin transport,strawberry was treated with auxin and other hormones to test the relationship between auxin and PHL gene expression.In addition,to characterize the expression patterns of these two genes,their expression levels in different organs and at various fruit development stages in citrus and strawberry were measured.Furthermore,to achive gene overexpression and RNAi-medaited gene silencing for these two genes,plant overexpression vector and RNAi vector were constructed respectively.This preliminary study aims to construct the genetic stocks that alter the expression of PHL and AHP genes in sweet orange and strawberry,which will ultimately lead to functional dissection of these two genes in the control of fruit acidity and sugar/acid ratios.The main results are summarized below: 1.Expression analysis and hormone response of FvPHL genesThe PH orthologous gene in strawberry belongs to a small gene family which includes 6 members,named FvPHL1~FvPHL6.These genes exhibit different but overlapping expression patterns.The expression difference of FvPHL1/2/6 in roots,leaves,flowers and fruits is not significant.PHL3/4/5 are preferentially expressed in one or two organs;for example,FvPHL4 is strongly expressed in leaf and flower with much lower expression in root and fruit.In contrast,FvPHL5 is strongly expressed in root while its expression in the other organs is more than 10-fold lower.The expression of FvPHL2/3/6 in fruit is relatively high,and among these,FvPHL3 is the most fruit-preferentially expressed genes,with a more than 180-fold difference compared to roots and fourfold higher expression than that in flowers.In addition,FvPHL genes exhibit fruit development and ripening-related expression patterns.We subjected the two-week-old diploid strawberry Yellow Wonder 5AF7 seedlings to the auxin treatments with different doses of IAA,0.01,0.1,1 and 10 ?M,for 24 hrs.Our results showed that all the other four FvPHL genes were up-regulated besdies FvPHL2 and FvPHL4.The strawberry fruits were also treated with the other four hormones,abscisic acid(ABA),6-benzyl amino adenine(BA),ethephon(ACC),and gibberellin(GA3).The results showed that BA could slightly regulate the expression of FvPHL3/5/6,and ACC and GA3 could regulate the expression of FvPHL5.ABA can affect the expression of six FvPHL genes.Exogenous hormones IAA and ABA seemed to have no effect on the expression of FvPHL genes in developing fruits.2.Expression patterns of PH and AHP5 genes in sweet orange and strawberryThe expression of CsPHL1 gene and CsAHP5 gene in different tissues and fruits of a sweet orange variety Newhall was analyzed by qRT-PCR.CsPHL1 gene is widely expressed in Newhall.The expression of CsPHL1 gene in leaves and flowers is higher than that in other organs.The expression of CsPHL1 gene increases gradually with fruit development,although the magnitude of change is not very big.The expression of CsAHP5 gene was the highest in the flower of Newhall.With the fruit development,the expression of CsAHP5 gene increased significantly,and was substantially higher at the fruit ripening stage than other stages.Similarly,qRT-PCR was applied to analyze the expression of strawberry roots,leaves,flowers,fruits and fruits at different developmental stages(10 days past anthesis/DPA,15 DPA,20 DPA and 25 DPA).FvPHL1 gene was expressed in all organs of strawberry,and its expression in roots was 5~10 times higher than that in leaves and flowers.With the development of fruit,the expression level of the gene showed an increasing trend.3.Construction of plant expression vectors,genetic transformation and gene expression analysis of transgenic plantsThe plant expression vectors were constructed based on the pFGC5941 and pFGC5941 M plasmid.Vectors of overexpression and RNA interference(RNAi)-based gene knockdown were constructed for CsPHL1,CsAHP5,FvPHL1 and FvAHP5.CsPHL1 gene overexpression vector ZZ302 and RNAi vector ZZ305;CsAHP5 gene overexpression vector ZZ321 and RNAi vector ZZ320;FvPHL1 gene RNAi vector ZZ312;FvAHP5 gene RNAi vector ZZ325 and its modification ZZ325 K.The results of genetic transformation of citrus and strawberry showed that 18 ZZ302,15 ZZ305,7 ZZ321,and one ZZ325 K transgenic plants were obtained.qRT-PCR was used to analyze the expression of target genes in PCR-positive transgenic plants,with one transgenic line showing CsPHL1 gene overexpression and three lines exhibitng down-regulation CsPHL1 expression.