| Grape(Vitis vinifera L.)contains a lot of phenols such as procyanidins(PAs)and stilbenes.PAs,also known as condensed tannins,has the effects of eliminating human free radicals,preventing diabetes,cardiovascular diseases and other health functions.Stilbene compounds participate in the defense response of plants.They are not only an important phytoalexin,but also a kind of health care products.They have anti-cancer,anti-inflammatory,antioxidant,cardiovascular disease prevention.BS(BSISTER)transcription factor gene is a member of MADS box gene family.Previous studies have shown that BS genes are mainly involved in the regulation of proanthocyanidins(PAs)synthesis in seed coat,plant auxin signal transduction and seed development in model plants,but the target genes and regulation mechanism of BS transcription factors are not clear.In order to explore the function and mechanism of BS transcription factors in grapes,VviBS1,VviBS2 and VviBS3,which are homologous to Arabidopsis BS,were identified and cloned.The main results are as follows:PAS,also known as condensed tannins,is a flavonoid polymer,which has the effects of scavenging human free radicals,preventing diabetes and cardiovascular diseases.Stilbene compounds participate in plant defense reactions and are an important phytoalexin.At the same time,they have anti-cancer,anti-inflammatory and other health effects,1.Three BSs transcription factors were identified in grape genome and performed expression analyses,subcellular localization analyses and transcriptional activation activity analyses.The results showed that VviBS3 was only expressed in grape tendrils,while VviBS1 and VviBS2 were highly expressed in flower buds,flowers and ovules.The expression patterns of VviBS1 and VviBS2 were similar and showed a downward trend during ovule development.Subcellular localization analysis showed that they were located in the nucleus.Transcriptional activation activity analysis showed that VviBS2 had self activation activity in yeast.2.DAP-seq technology,transcriptome and phenolic metabolome analysis were used to analyse the function of VviBSs.DAP-seq technology was used to explore the downstream genes regulated by VviBS2.It was found that the genes that VviBS2 can directly participate in PAs synthesis,auxin signal transduction,ethylene biosynthesis,amino acid metabolism and lipid biosynthesis biological pathways.Results of joint analysis of transcriptome and phenolic metabolome showed that in VviBS1 and VviBS2 transgenic grape callus,the expression of stilbene synthase genes decreased significantly,the content of stilbene decreased accordingly;the content of flavanone metabolites increased,and the downstream flavonols also increased.The expression of ANR and LAR genes was up-regulated,and the contents of catechin,epicatechin and dimers were significantly increased.VviBS1 and VviBS2 transcription factors up regulate the metabolic synthesis of PAs,flavanones and flavonols,and down regulate the synthesis of stilbenes.3.VviBS1 and VviBS2 may promote procyanidin synthesis and change the components of PAS subunits.VviBS1 and VviBS2 were overexpressed in Arabidopsis tt16 mutant and grape callus.It was found that VviBS1 and VviBS2 could increase the content of PAs in transgenic Arabidopsis and grape callus.The composition of PAs in wild-type and transgenic grape callus was determined by HPLC.It was found that overexpression of VviBS1 and VviBS2 not only changed the total amount of PAs,but also affected the proportion of subunits in PAs.EC-E,EC-T and C-T which can not detected in WT and VviBS2 transgenic callus were detected in VviBS1 transgenic callus.And %C in VviBS1 and VviBS2 transgenic callus was also higher than that in WT.The relative contents of ECG-T,ECG-E,%G and %EC in wild-type callus were higher than those in VviBS1 and VviBS2 transgenic callus.There were also differences in the composition of PAs subunits and the expression patterns of PAs synthesis related genes between VviBS1 and VviBS2 transgenic callus.VviBS1 and VviBS2 may play different roles in PAs biosynthesis.4.VviBS1 and VviBS2 may promote procyanidin synthesis by directly promoting the expression of key enzyme gene Vvi ANR1,Vvi ANR2 and Vvi LAR1.Yeast one hybrid experiments,double luciferase experiments,and Ch IP-q PCR experiments showed that VviBS1 directly bound and regulated the promoter activity of Vvi ANR2,and VviBS2 directly bound and regulated the promoter activity of Vvi ANR1 and Vvi LAR1.Yeast two hybrid and Bi FC experiments showed that VviBS1 interacted with Vvi SEP1,Vvi SEP2,Vvi SEP3,Vvi SEP4,Vvi FUL1,Vvi FUL2,Vvi AGL6 a and Vvi AP1.VviBS2 interacted with Vvi SEP1,Vvi SEP2,Vvi SEP3,Vvi SEP4,Vvi FUL1,Vvi FUL2,Vvi AGL6 a,Vvi AGL6 b,Vvi AP1 and Vvi AGL97.Double luciferase experiments showed that only Vvi SEP1,Vvi SEP3,Vvi AGL6 b and Vvi FUL1 could increase the activity of Vvi LAR1 promoter when injected alone and co injected with VviBS2.The expression patterns of Vvi SEP1 and Vvi SEP3 were similar to that of VviBS2.Transient expression of Vvi SEP1 and Vvi SEP3 alone or co injection with VviBS2 could increase the content of PAs in tobacco leaves.5.VviBS1 and VviBS2 might inhibite the synthesis of stilbenes by directly bind and repressing the expression of stilbene synthase gene Vvi STS48.The content of stilbene compounds in wild-type and VviBS1 or VviBS2 transgenic grape callus were analyzed by HPLC.The results showed that compared with wild-type grape callus,the content of piceid and resveratrol in VviBS1 or VviBS2 transgenic grape callus was lower than that in wild-type grape callus,which was consistent with the results of metabolome analyse.The STS expression level in VviBS1 or VviBS2 transgenic grape callus was lower compared with wild-type grape callus,which was consistent with the results of transcriptome analysis.Yeast one hybrid experiments,double luciferase experiments,and Ch IP-q PCR experiments showed that VviBS1 and VviBS2 inhibited the synthesis of stilbenes by directly bind and repressing the expression of stilbene synthase gene Vvi STS48.6.BS may regulate auxin signal transduction by directly regulating the expression of At IAA19 and At IAA29 genes,so as to regulate Arabidopsis thaliana root growth.After observing the root development of VviBS1 and VviBS2 transgenic tt16 lines,Arabidopsis tt16 mutant and wild type Arabidopsis Ws,it was found that overexpression of VviBS1 and VviBS2 in Arabidopsis could promote the growth of primary root and reduce the number of lateral roots,restore the response of Arabidopsis tt16 mutant to auxin.Meanwhile,it can also inhibit the expression of At ARF2,At ARF12,At ARF14,At ARF15,At ARF20,At GH,At GH3-2and At SAUR51 genes,and promote At IAA19 and At IAA29 genes expression.Double luciferase and yeast one hybrid experiment showed that BS can directly regulating the expression of At IAA19 and At IAA29 genes.In addition,overexpression of VviBS1 and VviBS2 in Arabidopsis can also increase seed germination rate,reduce etiolation of Arabidopsis seedlings,and inhibit ACS gene expression in seedlings. |
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