Construction Of RNAi Expression Victor Of PAL Gene In Seed Coat Of Cucurbita Pepo And Varieties Resistance To Plant Disease

Cucurbita pepo is an annual and sprawling herb was planted in a large areas of greenhouse innorth of China. Hull-less Cucurbita pepo is one of new cultivars of Cucurbita pepo, which is a raretype of variant in nature. However, the conventional breeding techniques are always be used inbreeding new cultivars and it have brought lots of questions. For example, conventional breedingtechniques are always difficult in breeding of new cultivars, and the new cultivars often hasunstable hull-less rate, low homozygosity, and it synthetic merchandise properties was not superiorto other varities. Meanwhile, the occurrence and damage of Podosphaera xanthii and virus diseaseof WMV are become more and more serious with the cultivated areas increased. It has beenconsidered as one of the restraining factors in hull-less Cucurbit pepo production. Therefore, hullCucurbita pepo and hull-less Cucurbita pepo were used as the material, and the RNAi expressionvector of PAL gene was constructed, and the location of PAL gene coding protein was predicted andobserved in this thesis. In addition, the relative expression of PAL gene in different tissues wasdetermined after inoculated with Podosphaera xanthii and WMV or infected by Podosphaeraxanthii and WMV. The main results are as follows:(1) EST sequences of PAL gene of hull Cucurbita pepo and hull-less Cucurbita pepo werecloned by the method of “Homologous recombination”. The sequence length was more than815bpand812bp, respectively. In addition, the EST sequence of PAL gene in both hull Cucurbita pepoand hull-less Cucurbita pepo were compared by the software of BLAST, compared with the PALgene sequences of EST in cucumber that have been reported in NCBI. The results showed that thesimilarity of PAL gene sequence of hull Cucurbita pepo and hull-less Cucurbita pepo to thereported PAL gene sequences of EST in cucumber were92.44%and92.76%, respectively.Meanwhile, part of target sequence in hull Cucurbita pepo PAL gene was used as the interferencefragment, and the specific primers were designed to amplify the sense and antisense fragments, andthe size of intron sequence of799bp form middle victor of pHANNIBAL was used to construct thestructure of hairpin, which was used to construct the RNA interference expression vector ofpCEPSPS-FPR (containing sense pal(f)-pdk intro-antisense pal(r)), and promoted by CaMV35Spromoter.(2) The cDNA fragments of PAL3and PAL13were used as the target gene to predict its codingprotein in subcellular organelles by using molecular biology software. The results showed that the encoding protein of PAL gene was located in cytoplasm and endoplasmic reticulum. Meanwhile, theconstructed vectors of pCAMIBA-PAL3-GFP and pCAMIBA-PAL13-GFP were transferred to onionepidermal cells by the method of “Biolistic bombardment”. Thereafter, the position of theexpression vector of pCAMIBA-PAL3-GFP and pCAMIBA-PAL13-GFP were located in cytoplasmand endoplasmic reticulum under the laser confocal microscopy. Therefore, the encoding protein ofPAL3and PAL13gene were located in cytoplasm and endoplasmic reticulum.(3) The relative expression of PAL gene was significantly improved after inoculated with thepathogen of Podosphaera xanthii in different resistant varieties of Cucurbita pepo in vitro. Also, therelative expression of PAL gene was significantly different from different tissues and differentvarieties after determined at different days. The relative expression of PAL gene increased indifferent tissues and different varieties with the inoculation days increased at initial stage, butdecreased in later. The relative expression of PAL gene in the leaves of disease-resistant varieties ofXiaosanxing F2and Sixing F1were significantly increased after inoculated with Podosphaeraxanthii, but the petiole and stem were slightly increased compared with the leaves. The maximumrelative expression quantity of PAL gene were at7and9days after inoculation in vitro; But therelative expression of PAL gene in the leaves of susceptible varieties of Jin12F2and Jin12F1waslower than the disease-resistant varieties of Xiaosanxing F2and Sixing F1after inoculated withPodosphaera xanthii, but the relative expression of PAL gene increased significantly, comparedwith the control. The maximum relative expression quantity was at9and11days. Therefore, therelative expression of PAL gene had a positive correlation with the disease resistance of varieties,and PAL gene is closely related to the disease resistance in Cucurbita pepo.(4) The relative expression of PAL gene was increased after inoculated with the pathogen ofWMV in different tissues and different resistant varieties of Cucurbita pepo at initial stage, butdecreased in later. Also, the relative expression of PAL gene in the leaves of differentdisease-resistant varieties of Cucurbita pepo was significantly higher than the tissues of petiole andstem after inoculated with WMV. Meanwhile, the relative expression of PAL gene in five differentvarieties was significantly different from the control after determined at different days, and therelative expression of PAL genes in resistant varieties and middle-resistant varieties were higherthan susceptible varieties. The relative expression of PAL gene in the resistant varieties of GBRV-8was higher than the middle-resistant varieties of GBRV-6, resistant varieties of GBRV-12andGBRV-13, and significantly higher than the susceptible varieties of Guangban. Therefore, relative expression of PAL gene had a positive correlation to the disease resistance of varieties.