Optimization Of VIGS System Using In Litchi Fruit And The Functional Characterization Of Some Key Genes In The Seed Development And Fruit Set Of Litchi

Litchi is famous for its fantastic fruit quality with fragrant sweet flesh and nutrition values.The seed abortion?chicken tongue?is a common inheritance trait in litchi.The seed abortion rate affects not only edible rate but also the taste and processing quality.Fruits with aborted seed display higher recovery rate and better taste.However,the interal mechanism of seed abortion in litchi is remained to be studied.Cell wall acid invertase plays an important role in the regulation of plant embryo development.Our previous study indicated that the low activities of cell wall acid invertase is the main cause for seed abortion in litchi cv.Nuomici.Two cell wall acid invertase genes?LcCWAI2 and LcCWAI5?and one cell wall acid invertase inhibitor gene?LcCIF?might closely related to the low enzyme activities.And furthermore,virus-induced gene silencing?VIGS?assay system suitable for litchi was set up and the critical role of LcCWAI5 in seed development was comfirmed.The success set up of litchi VIGS system provides a new way to validate the function of litchi genes.However,this VIGS assay system need to be further optimized and the function of LcCWAI2 and LcCIF are waiting for validation.In this study,we optimize the VIGS assay system suitable to induce the gene silence in litchi fruits.And this optimized assay system was applied to investigate the function of LcCWAI2 and LcCIF in seed development.The function of AP2/ERF genes in seed development and fruit setting were also explored for these kind of transcript factors were reported affecting the hexose to sucrose ratio and seed development in arabidopsis and rice.We investigated the effects of AP2/ERF silencing in litchi on the fruit setting and fruit development.Key AP2/ERF members affecting the fruit setting and fruit development were targeted base on RNA-Seq data.The main results were as follows:1.Tobacco rattle virus?TRV?is a suitable virus vector to induce gene silence with relative high efficiency and long lasting.The optimal infection Agrobacterium concentration is OD₆₀₀ around 1.0 for inflorescence dipping treatments.And the optimal treated period is at female blooming.Treated at three to five days before female blooming also showed the silencing effect with less efficiency,while treated after female blooming displayed no gene silencing effect.2.During female blooming and before 28 days after anthesis,the silence of LcCWAI5resulted in increases in the seed abortion rate in big seed and partly abortion cultivars and decreases in seed size,while the silence of LcCWAI2 only slightly reduced the seed size with no effect on seed abortion rate.Treated on 35 days after anthesis,the silence of LcCWAI5 showed no effect on seed development,while the silence of LcCWAI2 resulted in smaller seeds.These results suggested that LcCWAI5 is the critical cell wall acid invertase gene in determining the seed development and this gene played its role mainly in the early stage of seed development.Whereas,LcCWAI2 might play a certain role in affecting the mass accumulation of seeds especially during the mid and later stages of seed development.3.The expression of LcCIF in‘Nuomici'is much higher than those in big seed cultivar‘Heiye'and good endosperm developed cultivar‘Feizixiao'.The silence of LcCIF in‘Nuomici'resulted in better seed development and reduced abortion rate.These resutls indicate that LcCIF is the negative factor in litchi seed development.The activities of cell wall acid invertase in the funicle is significantly correlated to the expressions of LcCIF.This result suggested that the low cell wall acid invertase activities in‘Nuomici'might derive from the posttransciptional regulation of cell wall acid invertase inhibitor.4.RNA-Seq data demonstrated the higher LcCWAI5 expression in big seed cultivar‘Heiye'than seed-aborted cultivar‘Nuomici'at one week old fruits.There show obvious different in their promotor sequence.The relationship between the seqence difference and expression levels remain to be studied.5.The silence of AP2/ERF using VIGS resulted in higher fruit setting,bigger fruit size and lower hexose/sucrose ratio,but displayed no effects on seed development and aril sugar levels.Seven putative key AP2/ERF genes relating to fruit setting and fruit development were targeted base on RNA-Seq and their bioinformtions were analysis.