Construction Of The Plant Expression Vector Of Tomato CRY1 And CRY2 And Agrobacterium Tumefaciens-mediated Transformation

The functional analysis of cryptochrome gene family is mainly conducted in theArobidopsis. There are two publications about the function of cryptochrome familyin tomato so far. One is about the CCT1 antisense transgenic plants. The progenyfrom this transformant showed an elongated hypocotyl under blue light not under redlight. And the synthesis of anthocyanins under blue light was reduced in antisensetransgenic seedlings. In contrast, carotenoid and chlorophyll levels and secondpositive phototropic curvature were essentially unaltered. Another paper is about theoverexpression of CRY2. The phenotypes of tomato CRY2 overexpressor includeinternode shortening under both low-and high-fluence blue light, andoverproduction of anthocyanins and chlorophyll in leaves and of flavonoids andlycopene in fruits. CRY2 overexpression causes an unexpected delay in flowering,observed under both short- and long-day conditions, and an increased outgrowth ofaxillary branches. In this paper, we construct the plant expression vectors withtomato CRY1 and CRY2, and transfer the vectors into tomato through Agrobacteriumtumefaciens EHA105. We intend to elucidate relationship between cryptochromegene function and lycopene accumulation.There are two parts of the current research. First part is about the constructionplant expression vector. We construct five plant expression vectors, includingpBI121fsp-CRY1RNAi, pHB-CRY1RNAi, pHB-CRY10X, pHB-CRY2RNAi,pBI121fsp-CRY2OX. We extracted the total RNA of tomato, then obtained thecDNA. We got the PCR products to construct the vectors. We used two differentplant expression vectors. One is pHB with CaMV35s promoter and the other ispBI121fsp with fruit specific Lefsml's promoter. The resultant expression vectors are named aspBI121fsp-CRY1RNAi, pHB-CRY1RNAi, pHB-CRY10X, pHB-CRY2RNAi,pBI121fsp-CRY2OX. All of them were transformed into Agrobacterium tumefaciensEHA105 respectively. Restriction endonuclease and PCR tests comfirmed thatplasmid pBI121fsp-CRY1RNAi, pHB-CRY1RNAi, pHB-CRY10X, pHB-CRY2RNAi,pBI121fsp-CRY2OX were constructed successfully and the gene were integratedcorrectly in the vector.The second part is the tomato transformation mediated by Agrobacteriumcontaining the plant expression vector derived from CRY1 and CRY2. Tomato seedswere sterilized by 10% sodium hypochlorite solution and germinated in growthchamber. The excised cotyledons after 6-8 days germination were pre-cultured inpre-incubation medium. Agrobacterium tumefaciens was grown in LB liquidmedium supplemented with 20mg/L rifamycin and 100mg/L kanamycin, incubatedovernight at 28℃with shaking 200rpm. The explants were co-incubated withAgrobacterium tume faciens containing the expression vectors. The explants werethen transferred onto selective differentiation media with the correspondingkanamycin (50mg/L) and Basra(2mg/L), respectively. At last, the transgenic plantswere detected by resistant markers. The results indicated that the transgenic tomatoplants were obtained successfully.