| Tomato (Lycopersicon esculentem.Mill) have been widely planted over the world, due to its rich nutrition, delicious flavor, broad adaptability and high yield. Moreover, it is one main kind of vegetable crop in people’s daily lives. Fruits of mutant tomato are rich in lycopene used in oxidation resistance, delaying senescence, and immunity enhancement and anticancer. Therefore, the edibleness and economic value of lycopene has attracted much attention and become one of the hot spots of the international food and nutrition indicators of functional studies. However, it is still in the primary stage in China. In order to increase matched the tomato harvesting time and register day in the market and reduce damage rate in the process of transportation, breeding storable tomato is the major goal of matched tomato in recent years. However, the lycopene in the fruit of the storable tomato is general low, and this storable tomato couldn’t be effectively chosen. With the rapid development of molecular biology and genomics, the recombinant DNA technology; improving crop quality by genetic engineering has become the main efficient way to breeding crop. In recent years, researchers have found much single gene mutants on which the plant light metabolism could be affected. For instance, high-pigment hp mutants in tomato could increase carotenoid content, especially to the lycopene content. Consequently, the tomatos with high-pigment hp mutants have attracted much attention for numerous breeders and researchers. Presently, the discovered mutant hp1, hp2and hp3of the hp class evidently impact the lycopene content. Due to its general low lycopene content, storable tomato fruits become the material weakness on quality requirement of the current and future business and the market. One of the major problems eager to be solved is that matched tomato with high lycopene needs quick and efficient breeding. However, reports related to the transformation of storable tomato are limited; as a result, much inconvenience has been caused in the quality molecule breeding of matched tomato.Therefore, in this study, I compared the7mutants that contain3different hp genes(hp1, hp2and hp3) introduced from the Genetic Research Institute in the United States and the high pigment cultivated tomato; analyzed the lycopene and quality differences and other related elements; chose one of the3different hp genes which played the leading role to the lycopene and contains other fine quality as the donor of the matched tomato hp transgene; constructed and optimized the regeneration system and genetic transformation system for matched tomato; cloned the full-length of the target gene (hp2) and constructed the entry vector pCAMBIA-1301which contained the attR recombination sites; guided the target genes (hp2) into the matched tomato through the method of Agrobacterium-mediated to get the transgenic plants and tested and analyzed their molecular and physiological indicators to offer theoretical basis for the application of hp genes in matched tomato and create new germplasm for the tomato in high lycopene content. The main results are as follows:(1) Tomatoes with3different types of mutations in hp genes and without hp genes were chosen as materials. The analysis of the significant and extremely significant differences between the lycopene content in tomato fruits with different hp gene showed:Its content trend was hp2>hpl>hp3> non-hp, in which the hp2gene played an important role on the content of lycopene in the tomato fruits, and other major qualities of the tomato fruits are fine.(2) Tomatoes with3different types mutations in hp genes and without hp genes were chosen as materials. The analysis of the lycopene content and other main qualities in tomato fruits showed: there was no significant correlation between lycopene content in tomato fruits with different hp genes and other five major fruit quality indicators. The trend of both showed positive correlation and the trend was the size of sugar-acid ratio> Vitamin C content> soluble protein content> content of total soluble sugar> acidity content.(3) hp2gene mutation types of LA3006and LA4013in tomato were chosen as materials. The analysis of the gene cloning and constructing vector by RT-PCR method showed:hp2gene contained the attB sequence could be successfully obtained in tomatoes LA3006and LA4013, and constructed the entry carrier group sites with the ccdB gene which was named as pCAMBIA-1301-ccdB and double element expression vectors containing hp2gene which was named as pCAMBIA-1301-hp2.(4) The matched tomato varieties08076and09836were chosen as materials. Through the high frequency regeneration of cotyledons, the differential analysis of the main effect factors on the regeneration system showed:in the high frequency regeneration of cotyledon system of the storable tomato, different tomato cultivars had different extremely significant differences only in the bud number and differences between other induction and differentiation factor indicators aren’t significant. The cotyledons optimal high frequency regeneration system of the storable tomato included the culture of aseptic seedlings, seed disinfection by soaking with10%NaClO15min, MS culture medium (import,2.215g/L)+0.70%agar+2%sucrose; aseptic seedling culture medium with+1%sucrose1/2MS+0.7%agar; callus induction culture medium is MS+6-BA1mg/L+IAA0.2mg/L; adventitious bud differentiation culture medium is MS+6-BA1.5mg/L+IAA0.75mg/L; MS+IBA0.25mg/L medium for rooting, the storable tomato regeneration frequency are09836and08076of the cotyledon differentiation rate were100%and98.25%, respectively.(5) The matched type tomato varieties09836were chosen as materials. Through the Agrobacterium mediated genetic transformation method, analysis showed:the hp2gene could be used to import, storage type tomato09836, among them in the co-culture period, survival169, the survival rate was42%; in the adventitious bud differentiation period, the survival of65don’t shoot, survival rate of38%; in the seedling period, survival38, the survival rate was58%; the training period in resistant plants, finally get to14km resistant plantlets, the survival rate was36%.(6) Resistant plants obtaining from the genetic transformation were chosen as materials, the analysis of validation of the "purpose of PCR","NPTII gene" and "semi-quantitative RT-PCR" showed:there were8represented positive plants, respectively T0-01,T0-02,T0-05,T0-06,T0-08,T0-10,T0-12,T0-14,whose positive rate of transformed strains is57.14%.(7) The obtained positive plants were chosen as materials. The analysis of variance and multiple comparisons showed:the content of lycopene, chlorophyll a, chlorophyll b and carotenoid were significantly improved, of which content of lycopene in fruit respectively increased by309.64%-1571.08%; content of chlorophyll a respectively increased the139.45%-295.11%, that of chlorophyll b respectively improved152.58%-376.29%, content of carotenoid in leaves respectively increased by20.32%-95.72%.(8) The obtained positive plants were chosen as materials, the correlation analysis which content of lycopene in fruit of positive plant compared with contents of chlorophyll a, b and carotenoid in leaves showed that To generation of positive strains lycopene in fruit is significant positive correlation with content of chlorophyll a and carotenoid in their leaves, while content of chlorophyll b was positively significant correlation (r=0.79); there was a significant positive related among content of chlorophylla,chlorophyll b and carotenoid in positive strains.(9) By transgenic technology, created three T2generation positive transgenic matched type tomato (09836), the fruit lycopene content of T2-1, T2-5and T2-10were33.9,83.7,94.6mg/kg, eopared with non transgenic tomato were increased by3.8-10.6times; the ASI of fruit is respectively2.1,1.9and2.3, was decreased by45.24%-54.76%; the shelf life of fruit were48,42and53days, compared with the control was decreased by27.4%-42.47%; the hardness of fruit are respectively3.35kg/cm2,3.02kg/cm2and4.05kg/cm2, compared with the control was decreased by27.68%-46.07%. |
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