Cloning And Function Analysis Of Genes Belonged To SHINE Transcription Factor Family Associated With Navel Orange Wax Formation

Glossy degree of navel orange peel is an important factor determining navel orange fruit exterior quality.The successful breeding of "Ganqi 3" navel orange with glossy fruit peel provides a good material for studying the synthesis and transport molecular mechanism of citrus cuticular waxes.Three genes belonged to SHINE transcription factors family,named CsSHN1,CsSHN2,CsSHN3 were cloned from Newhall navel orange in this study.At the same time,real-time quantitative PCR were used to detect the time and space express difference of three CsSHNs between Newhall and "Ganqi 3" navel orange.Finally,in order to provide new genetic resources and theoretical basis for improving navel orange exterior quality by genetic engineering technology,the role of CsSHNs in the process of navel orange cuticular wax's synthesis was validated by transgenic technology.The results of the study are as follows:(1)Three genes belonged to SHINE transcription factors family,named CsSHN1,CsSHN2,CsSHN3,were cloned from Newhall navel orange in this study.The full length of CsSHN1 cDNA sequence is 608 bp and contains a 588 bp open reading frame,encoding 195 amino acids.The cDNA sequence of CsSHN2 gene is 619 bp and contains a 609 bp open reading frame,encoding 202 amino acids.The CsSHN3 gene is 664 bp in length and contains a 654 bp open reading frame,encoding 217 amino acids.The N end of the three proteins contain a AP2 domain.(2)Real-time quantitative PCR revealed that the three SHINE family genes were expressed in stem,leaf,flower and fruit of Newhall and “Ganqi 3”.The highest expression levels of these genes were detected in flowers,followed by leaf,peel and stems.In flower tissues,the CsSHNs were highest expressed in pistil,followed by stamens,petals and calyx.The expression levels of CsSHNs was increasing in the process of flower and fruit development,.Furthermore,the expression levels of CsSHNs in all tissues of "GanQi 3" navel orange were always lower than that in Newhall navel.(3)The overexpression vectors of CsSHNs were constructed and transformed to Arabidopsis through Agrobacterium tumefaciens mediated inflorescence infection method.The experiment of TB dying and chlorophyll permeability showed that the permeability of transgenic plants were decreased compared with wild-type Arabidopsis.Scanning microscopy(SEM)results showed that the wax crystals on the surface of transgenic plants stems and leaves increased significantly compared with wild-type Arabidopsis.Real-time quantitative PCR revealed that the expression levels of CsSHNs downstream genes,including AtCER1?AtCER3 and AtKCS1,were increased in transgenic plants.In summary,our results revealed that the overexpression of CsSHNs led to the upregulation of CsSHNs downstream wax related genes,which cause the increase of wax crystals on stems and leaves of transgenic Arabidopsis.Thus,we confirmed that CsSHNsgenes play an important role in citrus wax synthesis.