Expression Analyses And Functional Verification Of Proanthocyanidin Precursor Transmenbrane Genes MATE And GST In Persimmon Fruit

Persimmon fruits accumulate a large amount of proanthocyanidns(PAs) in the vacuole during the development stage, which cause a strong astringency sensation. The precursors of PAs is sythesized in cytosol, and then transported into vacuole and initialed polymerizaiton. Glutathione-S-transferase(GST) and Multidruge and Toxic Extrusion(MATE) transpotor were characterized as vacuolar transporter. The role of GST or MATE transporter in persimmon are not defined.In this work, Chinese PCNA type ‘Luotian Tianshi’, non-PCNA types(‘Mopanshi’) and Japanese PCNA(‘Youhou’) were used to investigate the changes of soluble, insoluble, total PAs content in different development stage. Expression patterns of Dk GSTL, Dk MATE1 gene at different developmental stages and various organs of different types deastringent persimmon were carried out to detect the function about these two gene mediate PAs biosynthesis. Moreover, constrcution of over-expression and interference Dk GSTL, Dk MATE1 gene in ‘Mopanshi’, ‘Gongcheng Shuishi’, and ‘Jirou’ transgenetic linese were through leaf disc transformation metheod. After PCR amplification and and q RT-PCR of PAs biosynthesis pathway related genes in transgenic materials and wild genotypes, we regarded that the Dk GSTL and Dk MATE1 may participate in the PAs biosynthesis pathway in persimmon. The main results are as follows:1) Changes of PAs relative concentration and content in persimmon fruit at different developmental stages of different astringent types. Soluble, insoluble PAs concentration were highest at 2.5 WAB and then decreased with fruit enlarge. Soluble PAs concentration in ‘Youhou’ fruit decreased to 0.2% at 10 WAB. However, soluble PAs in ‘Luotian Tianshi’ and ‘Mopanshi’ fruit are always maintain at a relatively high level. At 25 WAB, fruits of ‘Luotian Tianshi’ could remove astringency naturally, while soluble PAs contents and concentration of ‘Mopanshi’ are still high which meant ‘Mopanshi’ could not lose astringency naturallly. Insoluble PAs concentration of ‘Luotian Tianshi’ fruit increased between 10 WAB and 15 WAB with a peak compared with ‘Youhou’.2) Expression of Dk MATE1 and Dk GSTL gene at developmental stages and organs of different de-astringent types. Dk MATE1 showed highest expression in leaves, whereas Dk GST in carlyx, which suggested both genes were not specific expression in fruits. The expression of Dk MATE1 increased between 10 WAB and 15 WAB in ‘Luotian Tianshi’. Meanwhile, Dk MATE1 in ‘Youhou’ showed a considerable lower expression than ‘Luotian Tianshi’. It is suggested that Dk MATE1 transporter might have a preference for PAs precursors, which resulted in differences between C-PCNA and J-PCNA PAs structure.3) Function identification of Dk MATE1 and Dk GSTL. Overexpression or interference of Dk MATE1 and Dk GSTL transgenic lines were generated by using Agrobacterium-mediated genetic transformation in the ‘Mopanshi’, ‘Gongcheng Shuishi’ and ‘Jirou’. Positive rate of transgenic plants is 3.41% to 9.49%. Expression level of PAs biosynthesis pathway genes in transgenic material showed remarkable impacts of Dk MATE1, Dk GSTL on Dk ANR, Dk MYB4, Dk PA1. Expression of Dk ANR, Dk MYB4 and Dk PA1 tended toward up-regulation in transgenic plants of overexpression of Dk MATE1 and Dk GSTL. While for interfere lines, Dk ANR, Dk MYB4 and Dk PA1 are of remarkably down regulation. PAs precursors in cytoplasm had feedback regulation for PAs biosynthesis pathway upstream gene. Dk MATE1 and Dk GSTL involved in PAs regulation.4) Effects of ZT concentration on regeneration of ‘Gongcheng Shuishi’ and ‘Jirou’ leaves. DKW medium + ZT 4.0 mg /L + IAA 0.1 mg /L is suitable for persimmon adventitious buds regeneration within 2-3 months, and the regeneration rate is up to133.33%.