Functional Characteriztion Of NL32 And PGA4 Involved In Ascorbate Accumulation In Tomato

Ascorbic acid(AsA)is an antioxidant that plays a very important role;it is also essential in a variety of physiological process of plant.In humans,ascorbic acid plays an irreplaceable role in maintaining normal function of human body,and humans do not have the ability of ascorbic acid synthesis by themslf.So they obtain the required ascorbic acid only from the dietary scuorces.Fresh fruits and vegetables serve as the main scource of AsA,and tomato is the major vegetable in the human diet that has high content of ascorbic acid.Therefore,it is important to study how to improve the content of ascorbic acid in tomato.Now in tomato,several key genes in ascorbic acid synthesis and metabolic pathway have been cloned,but the regulation mechanism ascorbic acid accumulation remains to be the further studied.In this study,we investigated the correlation between the content of ascorbic acid and some leadSNPs by GWAS,and we selected 6 candidate genes in the range of 50 Kb.By studying the gene expression levels,two genes were further selected for transgenic functional verification.The main results of this study are as follows:1.We obtained several candidate segments on chromosome 6 and chromosome 9.In addition,we chose 6 candidate genes:Solyc06g009200,Solyc09g064680,Solyc09g064 6 70,Solyc09g066290,Solyc09g065810 and Solyc09g065820.2.By Q-PCR,the relative expression of seven genes in tomato in relation to ascorbic acid content was analysed,which showed that Solyc06g009200 and Solyc09g064680 expression and ascorbic acid content is a linear relationship.Solyc06g009200 was lowly expressed in high-AsA tomato,but highly expressed in low-AsA tomato.The expression pattern of Solyc09g064680 was contrary of the Solyc06g009200.For the expression of the other 4 genes,there is no obvious connection between gene expression and AsA content.3.According to the genetic structure analysis,Solyc06g009200(PGA4)is a polymer galacturonic acid enzyme;Solyc09g064680(NL32)is a member of NL subtribes of NBS-LRR family in tomato.4.In this study,we constructed SIPGA4 overexpression vector and SINL32 RNAi vector,then transferred them into genome of tomato variety Ts265 using the Agrobacterium-mediated transformation.The putative transgenic plants were identified by PCR,and we screend out some positive transgenic tomato.5.Analyzed the relative expression of target gene in transgenic plants,and the transcript levels of SlNL32 had been suppressed significantly in RNAi transgenic lines.The expression of SlPGA4 in over-expressing transgenic lines was significantly enhanced.6.We used the protocol by Gillespie to determine the ascorbic acid content.Ascorbic acid content in leaves and fruits of the transgenic plants was determined by ELISA.The results showed that ascorbic acid concent was significantly elevated in leaves and fruits of SlNL32 RNAi transgenic lines and ascorbic acid content was decreased significantly in leaves and fruits of SlPGA4 over-expressing lines compared with the control.7.Analyzed of expression of the known genes involved in major ascorbic acid synthesis and metabolic pathway in leaves and fruits of transgenic tomato.In the leaves of SlNL32 silenced lines,the expression of most of synthetic genes were increased,and the relative expression of cAPX was inhanced,but the expression of AO,tAPX and AOBP was significantly suppressed.The expression of most genes in D-mannose/L-galactose pathway were up-regulated,the expression of MIOX was significantly reduced compared with control in the red ripe fruits.In leaves of SlPGA4 over-expressing lines,except the PMI,MDHAR and DHAR,the expression of all the other synthetic genes was increased.For the oxidative genes,tAPX was suppressed,and the other three gene expression was increased.In the fruits,D-mannose/L-galactose pathway related genes such as GPI,PMI,GMP1,GMP2,GMP3 and GPP1 was transcriptionally increased,MIOX expression levels was significantly reduced.MDHAR expression was up-regulated and DHAR was down-regulated.cAPX and tAPX showed significant lower expression level,and AO and AOBP exhibited up-regulated expression.8.Treated SINL32 RNAi suppressed lines and Ts265 by growing seedlings in 1/2MS media supplemented with 10 μM MV and 100 mM NaCl,respesctively.The growth of the suppressed lines and Ts265 had been suppressed,but relative root length and plant height of the transgenic lines was significantly higher than the control.This result suggests that SINL32 suppression enhanced the tolerance of MV,but there is no significant difference in terms of NaCl.