Analyses Of Cloning And Expression Of The Genes Involved In The Resistance Disease From Two Chinese Wild Vitis Species

Grapevine is one of the most important fruits of economic crops in the world. In China,grape industry possesses very significant economic status. At present, cultivated areas andyields of grapevine rang the forefront in the world. However, powdery mildew (Erysiphenecator (Schw.) Burr.) and other pathogens restricted seriously the progress of grapevineindustry. Most cultivated Vitis vinifera worldwide with high quality fruits, were generallysusceptible to E. necator. China, as one of the most important origin centers of Vitis in theworld, possesses many species and diversity germplasm resource with the resistance topowdery mildew. Especially, Chinese wild Vitis pseudoreticulata becomes precious resistantand breeding resource, because of high resistance to pathogens. The results from our researchgroup showed that Chinese wild Vitis pseudoreticulata ‘Baihe-35-1’ displayed high resistanceto powdery mildew, so we constructed a full length cDNA library with the leaves induced byE. necator, and gained some transcription factors from the library to study their functions. Thepresent study mainly focused on the expression patterns of a novel WRKY transcription factorunder biotic and abiotic stresses, and its function analysis in transgenic tobacco plants byAgrobacterium-mediated transformation.Chinese wild Vitis quinquangularis ‘Danfeng-2’ possesses the resistance to powderymildew and has high content of resveratrol in ripe berries. Grape resveratrol, as a phytoalexin,can not only enhance plant resistance to various pathogens, but also play important roles inthe biological pharmacy industry, including anti-cancer, anti-tumor, prevention ofcardiovascular disease, and so on. Stilbene synthase plays a key part in resveratrolbiosynthesis pathway. Therefore, the specific-tissue expression of stilbene synthase family in‘Danfeng-2’ was discussed in order to obtain the stilbene synthase with high expression ofresveratrol.The main results are described as follows.1. Full length of the novel WRKY gene was cloned from Chinese wild Vitispseudoreticulata ‘Baihe-35-1’ using RACE method based on the constructed full lengthcDNA library, and was designated as VpWRKY3(Genbank Accession No. JF500755). Fulllength of VpWRKY3cDNA is1280bp and contains a complete open reading frame of960bpencoding a putative protein of319amino acids with a predicted protein molecular weight of 35.4kDa. Sequence analysis showed that VpWRKY3contains one WRKY domain, oneC2-HH zinc-finger motif (C-X5-C-X23-H-X1-H) and two predicted nuclear localization signal(RKRK, KKPR), and belongs to group II a of the WRKY superfamily. Transient expressionof VpWRKY3indicated that VpWRKY3protein is located in the nucleus of onion epidermalcells. Moreover, VpWRKY3can activate reporter gene expression in yeast and functioned as atranscriptional activator using yeast one hybrid. To identify the function on resistance diseaseof VpWRKY3, the expression assays of VpWRKY3were conducted by quantitative RT-PCR intwo grapevine genotypes including E. necator-resistant grapevine ‘Baihe-35-1’ and E.necator-susceptible ‘Baihei-35-2’. After infected with E. necator, VpWRKY3transcript wassignificantly induced until it reached the maximum level at12h, and then decreased in thetwo grapevine genotypes, which suggesting that VpWRKY3was involved in the response to E.necator in grapevine. Transgenic tobacco plants were obtained by Agrobacterium-mediatedtransformation and the results showed that VpWRKY3enhanced the resistance to pathogen intransgenic tobacco plants, as positive regulatory functions. In addition, SA, ABA and ethyleneinduced an increase of VpWRKY3expression in ‘Baihe-35-1’, and the maximum expressionlevel appeared at the8h, whereas MeJA was hardly induced, suggesting that VpWRKY3wasregulated by SA, ABA and ethylene, mainly. For under drought, high temperature, lowtemperature and osmotic stresses, the induced expression of VpWRKY3increased obviouslyand responded actively to abiotic stresses. Furthermore, VpWRKY3transgenic tobaccoseedlings might enhance the resistance to high salt by inducing formation of more lateralroots.2. Our research group cloned28members of STS family from Chinese wild Vitisquinquangularis ‘Danfeng-2’(Genbank Accession No. JQ868665, JQ868666, JQ868668,JQ868670-JQ868687, JQ868689-JQ868693, JQ868697, JQ868698), and we examined theexpression patterns of these genes in six different tissues of including young leaves, youngfruits, ripe fruits, fruit flesh, berry skins, and seeds by qRT-PCR. The results showed that28members of STS family from Chinese wild Vitis quinquangularis ‘Danfeng-2’, theirexpression in ripe fruits was higher than the other five tissues, followed by fruit flesh andseeds, and the expression in young leaves was the lowest. In fact, the expression level of themvaried in six different tissues of grapevine. Such as, the expression of VqSTS32(GenbankAccession No. JQ868666) was the highest in young leaf, that of VqSTS5(Genbank AccessionNo. JQ868693) in young fruit, VqSTS6(Genbank Accession No. JQ868692) in both ripe fruitsand fruit flesh，VqSTS25(Genbank Accession No. JQ868673) in skin, VqSTS2(GenbankAccession No. JQ868697) in seeds. In28members of STS family, the highest expression ofmost STS genes appeared in ripe fruits and fruit flesh, but there were some exceptions, such as the expression of VqSTS2(Genbank Accession No. JQ868697) and VqSTS5(GenbankAccession No. JQ868693) were both the highest in seeds. For fruit flesh, there was a verygreat distance of expression between the highest gene and the lowest gene, such as VqSTS6(Genbank Accession No. JQ868692was remarkably higher than VqSTS2(Genbank AccessionNo. JQ868697). Taken together, these results showed that the expression of VqSTS6among28members of STS family from Chinese wild Vitis quinquangularis ‘Danfeng-2’ was thehighest in six different tissues, which provided useful information for further study andapplication. Otherwise, by genomic walking method, two upstream regulatory sequences ofstilbene synthase (Genbank Accession No. JX537789and JX537790), were obtained fromChinese wild Vitis quinquangularis ‘Danfeng-2’. Sequences prediction analysis displayed thatthe two upstream regions have w-box related to disease-resistance and some othercis-regulatory elements associated with abiotic stress responses, which will be studied further.