Constrction Of CDNA Library And Analysis Of Expressed Sequence Tags Of Chinese Wild Vitis Pseudoreticulata Induced By Plasmopara Viticola

Grape downy mildew caused by plasmopara viticola,is one of the most seriously fungal disease of grapevine all over the world.All currently grown commercial grape cultivars(V.vinifera) are known to be susceptib to this fungal disease,previous reaserches indicated that Vitis Pseudoreticulata W.T.Wang,native to China,possessing resistance to downy mildew.The grape materials of Chinese wild V.pseudoreticulata clone Baihe-35-1 highly resistant to downy mildew were used in this study,plasmopara viticola inoculation was carried out under natural field conditions.The cDNA library was constructed utilizing SMART^TM cDNA Library Construction Kit.For understanding the genes expressed from this cDNA library in the disease-resistant interaction by EST sequencing and bioinformation analysis and providing the valuable disease-resistant genes for the breeding study on downy mildew resistance.Providing theory basis for molecular mechanism studies at the molecular level on disease-resistant interaction between Chinese wild Vitis and plasmopara viticola and Laying the ground-work of the EST analysis.1.Total RNA was extracted from inoculated leaves at 24h,48h,72h,96h respectively with SDS/phenol method modified partially,the cDNA library was constructed utilizing SMART^TM cDNA Library Construction Kit.The titer of primary library was about 0.92×10⁶ pfu/mL,of which 97%clones were recombinant,the library capacity was about 7.8×10⁵.The titer of amplified library was about 5×10⁹ pfu/mL,96%recombinants were obtained,and the inserted cDNA lengths ranged from 500bp to 2200bp.2.randomly chosing clones,extracting plamids and examinating the inserted cDNA lengths with PCR.The qualified clones(the inserted cDNA lengths were longer than 500bp, the consistency of plasmids were higher than 30ng/μL and A_260/280＞1.9,A_260/230＞2.0) were chosen to be sequenced from the 5′end of the cDNA.After removing the vector sequences, repeat sequences and shorter sequences(＜150bp),254 ESTs were acquired.The ESTs lengths mainly distribute from 500 to 600bp(27.06%),and the fragments that are longer than 500bp account for 64.17%,the longest sequence reaches 964bp,3.254 ESTs of 5' ends were obtained from the cDNA library.And all of them have submitted to GenBank database,the GenBank accession numbers are as follows,FG269201,FG269202,FG269203,FG269204,FG269205,FG269206,FG269207,FG269208,FG269209,FG269210,FG269211,FG269212,FG269213,FG269214,FG269215,FG269216,FG269217,FG269218,FG269219,FG269220,FG269221,FG269222,FG269223,FG269224,FG269225,FG269226,FG269227,FG269228,FG269229,FG269230,FG269231,FG269232,FG269233,FG269234,FG269235,FG269236,FG269237,FG269238,FG269239,FG269240,FG269241,FG269242,FG269243,FG269244,FG269245,FG269246,FG269247,FG269248,FG269249,FG269250,FG269251,FG269252,FG269253,FG269254,FG269255,FG269256,FG269257,FG269258,FG269259,FG269260,FG269261,FG269262,FG269263,FG269264,FG269265,FG269266,FG269267,FG269268,FG269269,FG269270,FG269271,FG269272,FG269273,FG269274,FG269275,FG269276,FG269277,FG269278,FG269279,FG269280,FG269281,FG269282,FG269283,FG269284,FG269285,FG269286,FG269287,FG269288,FG269289,FG269290,FG269291,FG269292,FG269293,FG269294,FG269295,FG269296,FG269297,FG269298,FG269299,FG269300,FG269301,FG269302,FG269303,FG269304,FG269305,FG269306,FG269307,FG269308,FG269309,FG269310,FG269311,FG269312,FG269313,FG269314,FG269315,FG269316,FG269317,FG269318,FG269319,FG269320,FG269321,FG269322,FG269323,FG269324,FG269325,FG269326,FG269327,FG269328,FG269329,FG269330,FG269331,FG269332,FG269333,FG269334,FG269335,FG269336,FG269337,FG269338,FG269339,FG269340,FG269341,FG269342,FG269343,FG269344,FG269345,FG269346,FG269347,FG269348,FG269349,FG269350,FG269351,FG269352,FG269353,FG269354,FG269355,FG269356,FG269357,FG269358,FG269359,FG269360,FG269361,FG269362,FG269363,FG269364,FG269365,FG269366,FG269367,FG269368,FG269369,FG269370,FG269371,FG269372,FG269373,FG269374,FG269375,FG269376,FG269377,FG269378,FG269379,FG269380,FG269381,FG269382,FG269383,FG269384,FG269385,FG269386,FG269387,FG269388,FG269389,FG269390,FG269391,FG269392,FG269393,FG269394,FG269395,FG269396,FG269397,FG269398,FG269399,FG269400,FG269401,FG269402,FG269403,FG269404,FG269305,FG269306,FG269307,FG269308,FG269309,FG269310,FG269311,FG269312,FG269313,FG269314,FG269315,FG269316,FG269317,FG269318,FG269319,FG269320,FG269321,FG269322,FG269323,FG269324,FG269325,FG269326, FG269327,FG269328,FG269329,FG269330,FG269331,FG269332,FG269333,FG269334,FG269335,FG269336,FG269337,FG269338,FG269339,FG269340,FG269341,FG269342,FG269343,FG269344,FG269345,FG269346,FG269347,FG269348,FG269449,FG369006,FG369007,FG369008,FG369009,FG3690104.ESTs similarity analysis based on BlastX software was finished by comparing sequences in GenBank,56.3%ESTs had higher homology with functional genes,14.6% ESTs(37 ESTs) had higher homology with hypothetical protein or unknown protein,and 20.9 %ESTs(53 ESTs) hitted no any sequences in GenBank.very possibly contains the Chinese wild Vitis unique disease-resistant related gene in these no-hits ESTs,after further analysis with BlastN and dbEST database.5.Most genes expressed with low abundance generated from the cDNA library and occupied 86%,indicating the library had a good complexity of gene composition.These ESTs with higher homology with functional genes may be classified into 10 different categories, the highest proportion of which was related to disease and defence(18.88%),and the others were energy and metabolism(16.78%),transcription(13.99%),signal transduction(13.29%),proteins synthesis(11.19%),protein destination and storage(4.89%),cell structure(4.89%),membrane and transport(4.20%),transposons(1.40%) and unclear classification(10.49%).We next step study is going to has the known function sequence to conduct the further deep research,and the massive unknown function sequences identification more important to the library,to clarify their function,to provide the effective gene for the disease-resistant breeding transgene research.