Promoter Analysis Of Cell Wall Invertase And Its Inhibitor In Litchi And Cloning Genes Of Key Genes In Longan

Litchi and longan both belong to the most famous four fruits in the south China.Their fruits have great edible value and fantastic nutritional value.Seed abortion?chicken tougue?is a common heredity phenomenon during fruit development of litchi.Seed size is one of the criteria for evaluating the quality of longon and litchi.In general,the flesh recovery rate of fruits with seed abortive are higher,and the quality are usually better.Traditional valuable litchi seed abortion litchi cultivars are‘Nuomici'and‘Guiwei'in Guangdong,and‘Lanzhu'in Fujian.There has no heritable cultivar with seed abortion in longan.But small seed of longan fruit is one important quality component and is taken seriously by the producers and researchers.However,the mechanism of seed abortion in litchi and longan is poor-documented with only structural,physiological and biochemical researches.The inner molecular mechanism of seed abortion is waiting for uncover.Previous studies of our research group found that cell wall invertase?CWIN?and its inhibitor played cruial role in the seed development of litchi.The abortion rate of big seed litchi cultivars increased in response to the silence of LcCWIN5 via virus-induced gene silencing assay,which comfirm the critical role of LcCWIN5 in the seed development.Similar to litchi,longan also belong to Sapindaceae family and is arilate fruit crop.The seed size of longan is an important factor in determining the fruit quality of longan.Is it CWIN and its inhibitor affect the seed development of longan?What is the inner mechanism of differential expression patterns of CWIN and its inhibitor genes among different seed size cultivars.These are problems waiting for answer.In the present study,in order to investigate the reason of differential expressions of CWIN and CIF among cultivars with different seed size in litchi,their promoter sequences were analyzed.In the longan,CWIN and its inhibitor genes were isolated from longan and their similarity to the genes from litchi and other plant species were analyzed.And the VIGS system of litchi was applied to longan to reduce seed size in of longan.During VIGS assay,we found pTRV2-Empty agrobacterium increase fruit drop.Thus the effects of agrobacterium on fruit thining of longan was also investigated in the present study.The main results were as follows:1.The upstream 2000 bp sequence LcCWIN2 and LcCWIN5 ORF were isolated from big seed cultivar‘Heiye',small seed cultivar,‘Feizixiao'and abortive seed cultivar‘Nuomici',respectively.The sequences of‘Nuomici'showed great difference from other cultivars after sequence alignment.These differences included endosperm relative or enhanced ciselement using the PlantCARE analysis,which might affect the activity of the promoters.2.The upstream 2000 bp sequences of LcCIF ORF were isolated from seven litchi cultivars differed in seed size.Two main differences could be found after sequence alignment:1)Some cultivars had a sequence of two repeats of 404 bp,and the other part contains only a 404 bp sequence.2)Two SNP found-1462 bp and-1870 bp.3.The LcCIF upstream sequence of‘Nuomici'differed with‘Heiye'and‘Feizixiao'in these two differences.In GUS assays,the LcCIF upstream sequence of‘Heiye'showed promoting activity by GUS qualitative detection and double fluorescent reporter gene detection,while that of‘Nuomici'did not show any activity.The 404 bp sequence was isolated from LcCIF and connected to the NOS combined-type promoter.Double fluorescent reporter gene detection showed with the increase of 404 bp sequence,and the NOS combined-type promoter activity decreased.4.Five full length of CWIN genes and one inhibitor CIF gene,named DlCWIN1,DlCWIN2,DlCWIN3,DlCWIN4,DlCWIN5 and DlCIF respectively were isolated from longan.Their sequence length are 1713 bp,1722 bp,1734 bp,1734 bp,1740 bp and 477 bp,and the amino acid number are 570,573,577,577,579 and 158.The target sequences were then subjected to bioinformatics analysis.Longan and litchi displayed high similarity of CWIN and the CIF sequences.5.The expression patterns of five DlCWINs and one DlCIF in different tissues and cultivars were determined by real-time quantitative PCR.DlCWIN1 and DlCIF were expressed mainly in mature leaves.DlCWIN2 was mainly expressed in the seed funicle,and seed coat of both cultivars and mature leaf of‘Shixia'.DlCWIN3 was expressed in the flowers and leaves.DlCWIN4 also showed high expression levels in flowers,and DlCWIN5were specifically expressed in the flower tiuuses.The expressions of DlCWIN5 in female flowers and male flowers were hundreds times higher than the expressions of other tissues.Among the six genes tested,the expressions of DlCWIN1 and DlCWIN2 generally increased,while the rest three CWIN genes decreased with fruit devleopment.The expression of DlCIF are low during fruit development except for a peak appeared at fifteen days after anthesis.6.Silencing DlCWIN5 by VIGS through injection at 21-30 days after anthesis displayed no obvious effect on seed size in cultivar‘Shixia'and‘Dawuyan',while a significant reduce seed weight were noticed in cultivar‘Shuangmaomu'.The silence of DlCWIN5 at female flower anthesis reduce the seed size of‘Chuliang'to some extent,but did not reach significant level.These results indicated that VIGS sassay system was not efficency in longan as comparing with litchi or DlCWIN5 was not the crucial gene in determining the seed development of longan.7.Since pTRV2-Empty agrobacterium(OD₆₀₀=1.0)treatment enhance fruit drop of longan cultivar‘Chuliang'.The effects of different concentration of agrobacterium on fruit thing of longan were then investigated.Different concentration agrobacterium treatments demonstrated similar thining effects,with around 30%reduction of fruit setting.Paralleling with reduction of fruit setting,OD₆₀₀=1.0 and 1.5 treatments increased fruit size and total soluble solid,while OD₆₀₀=2.0 displayed opposite effects.These results suggested that OD₆₀₀=1.0 to 1.5 might be the optimal concentration of agrobacterium in longan thining through biotic stress.