Construction Of Cerasus Humilis CCD1 Gene Plant Expression Vector And Genetic Transformation Of Tomato

Cerasus humilis is a unique shrub fruit tree in China.which is rich in calcium oleate,and known as "calcium fruit." The fruit has strong resistance,strong root system,strong ability to absorb water and nutrients,also can be used as windproof sand fixing tree species.Cerasus humilis fruit is bright in color,unique in taste,and rich in nutrition.which has high economic value and research value.To explore the metabolism of Cerasus humilis seze and flavor for future fruit color breeding and quality improvement laid a foundation.Under the catalytic cracking of the CCDs gene,carotenoids can produce deregulated carotenoids.which have many biological functions.The deregulated carotenoids produced by CCD1 and CCD4 cracking mainly participate in the color and flavor of flowers and fruits.formation.The previous study of this group showed that the Cerasus humilis CCD1 gene can crack lycopene to6-methyl-5-heptene-2 ketone;Beta-carotene can also be cracked to form ?-violet ketone.In this study,plant expression vector was constructed,and the Cerasus humilis CCD1 gene was introduced into tomato plants by the method of agricultural Bacillus.In order to induce gene expression,the function of the Cerasus humilis CCD1 gene was verified by color aberration analysis and determination of lycopene in tomato fruit.The main research contents are as follows: The plant expression carrier PBI121-CCD1 was constructed by PCR amplification and Gene fragment recovery technology.Connect the gene fragment to the PBI121 carrier fragment.The proposed plant expression vector was introduced into the EHA 105 by freezing and melting method.The main research contents are as follows:1.The complete sequence of Cerasus humilis CCD1 c DNA was amplified based on the sequence design primers of the CCD1 gene sequence and the plant expression carrier PBI121.PCR amplification restores the target gene fragment and connects it with the PBI121 plasmid carrier fragment to construct the plant expression vector PBI121-CCD1.The plant expression carrier PBI121-CCD1 was transferred to Agribacillus EHA105 by freezing and melting method.2.Based on the tomato regeneration system,the cotyledons of Micro-Tom Tomato were infected withagaric bacteria containing the target gene.The infection conditions include: infection concentration,infection time,co-culture time and antibiotic concentration.The optimal genetic transformation system was obtained by orthogonal design: the infection concentration of Agrobacterium was OD600 = 0.5,the infection time was 10 min,the total culture time was 1D,and the antibiotic concentration was 300 mg/L.The final adventitious Bud differentiation medium is: MS + ZT 2 mg/L + Timentin 300 mg/L + Kan 100 mg/L p H is5.8.Finally,the transgenic tomato plants were obtained by rooting and refining seedlings.3.The DNA of control and transgenic plants was extracted by CTAB method.The DNA of tomato was used as template for PCR verification.According to the results of gel electrophoresis,it can be preliminarily indicated that the CCD1 gene of Cerasus humilis can be transferred into tomato genome.Through the analysis of color difference of tomato fruit and the determination of lycopene content,it can be inferred that the protein encoded by Cerasus humilis CCD1 gene can degrade lycopene.