Preliminary Study On The Molecular Mechanism Of Light In Regulating Anthocyanin Biosynthesis In Pericarp Of Litchi Chinensis

As we all known,litchi(Litchi chinensis Sonn.)is an evergreen tree of the genus Litchi of the Sapindaceae family.It is an important cultivated fruit tree in southern China.Color is an important component of litchi fruit quality.Anthocyanins,which are biosynthesized through the flavonoid pathway,are the main pigments that determine the color of litchi fruits.Light is one of the key factors affecting the coloration and anthocyanin biosynthesis of litchi pericarp.The investigation of the molecular mechanism of light in regulating anthocyanin biosynthesis in litchi pericarp can provide theoretical basis for the appearance quality improvement of litchi fruit,and it has practical significance for increasing the commercial value of litchi.In the study,a representative litchi cultivar‘Guiwei’ with good coloring was used as the test material.Using RT-PCR,yeast double hybrids,bimolecular fluorescence complementation(BiFC),transient expression,double fluorescence reporting,and yeast single hybrids and other technical methods,the content of anthocyanins and the major enzyme genes in the pericarps after bagging and bag removal were analyzed.The key photoreceptor genes LcUVR8 and the key light regulatory factors LcCOP1 and LcHY5 were screened and cloned,and then sequence analysis and interaction analysis were performed.The activation ability of the key light regulatory factor LcHY5 on the structural gene promoter was determined.This study provides the primary regulatory network of light on anthocyanin biosynthesis in litchi pericarp.The main results are as follows:1.The black non-woven bag could obviously inhibit the accumulation of anthocyanins in the pericarp of ‘Guwei’ litchi.The bagged fruits had a pale-yellow color but could mature normally.Anthocyanins were rapidly biosynthesized after bag revomal,but the anthocyanin content was still lower than that in the control when matured.2.Real-Time PCR analysis of the expression of eight important structural genes in the anthocyanin biosynthetic pathway of the pericarp after bagging and bag removal found that bagging treatment significantly inhibited the expression of LcPAL,LcCHI,LcCHS,LcF3 H,LcF3’H,LcDFR,LcANS and LcUFGT.The expression of these genes increased after bag removal,among which LcPAL,LcCHI,LcCHS,LcF3H,LcDFR and LcANS reached their peak expression levels at 12 h after bag removal,and the expression patterns of LcF3’H and LcUFGT were basically consistent with the change of anthocyanin content.3.Real-Time PCR analysis of the expression of light receptors and light signal-regulated genes after bag removal found that LcHY5,LcCOP1,LcCOP10,and LcUVR8 were involved in light-regulating anthocyanin biosynthesis in litchi pericarp.The full length was these genes were cloned,which is 504 bp,1920 bp,519 bp,1437 bp,respectively,and the number of encoded amino acids is 167,639,172,and 478,respectively.4.Through yeast two-hybrid system and BiFC system,it was proved that there was interaction between LcUVR8,LcHY5,LcMYB1 and LcCOP1,but LcUVR8,LcHY5,LcMYB1 couldn’t interact with LcCOP10.5.The Agrobacterium tumefaciens-mediated transformation system confirmed that LcCOP1 could achieve functional complementarity with the Arabidopsis cop1-4 mutant.LcCOP1 negatively regulated anthocyanin biosynthesis,and restored the elongation of the hypocotyl of the mutant.Using the leaf transient expression system of transgenic K326 tobacco,it was proved that LcCOP1 could inhibit the expression of anthocyanin biosynthetic structure gene and inhibit the accumulation of anthocyanin in tobacco leaves expressing LcMYB1+bHLH3 gene.6.Analyzing the promoter region of important structural genes in the anthocyanin biosynthetic pathway,it was found that the promoter region of LcPAL,LcCHS,LcCHI,LcF3H,LcDFR,and LcANS had the LcHY5 binding element G-box.Using the ‘Guiwei’litchi genomic DNA as a template,the promoter region of the target structural gene was cloned.7.Using yeast one-hybrid system and dual luciferase assay,it was concluded that LcHY5 could activate the activities of the structural genes LcCHS,LcF3 H,and LcANS promoters.