Transgenic Technology System Research Of Litchi

Transgenic breeding technology has provided new direction for litchi breeding for it can not only speed up the breeding process but also improve targeted traits. The establishing and optimizing of transgenic technology system, searching and identifying functional gene as well as the expression and regulatory pattern of its promoter region are the key points of biotechnological breeding application. This study established the agrobacterium mediated transformation technology system using Feizixiao litchi embryonic callus as explant as well as the transgenic technology system using pollen tube channel to import litchi inflorescence. This study also cloned the litchi TLP gene promoter and constructed a series of plant expression vectors and then verified its expression pattern through transient expression method. The main results were as follows:1、Explore the optimum condition for agrobacterium mediated transformation system using Feizixiao embryonic callus as explant. The results show that:the infection ability of agrobacterium EHA105is better than LBA4404; microbial suspension till OD600=0.6, infect for30min, and co-culture for2days at25℃will get the best transformation effect; final concentration of100μmol/l AS can effectively increase the agrobacterium mediated transformation. The combination of hygromycin(40mg/1) and carbenicillin(700mg/1) is appropriate for screening resistant callus.2、Knowing the accurate time for litchi flower’s pollen tube channel to arrive at embryo sac is the key premise for using the pollen tube channel method. Result shows that about20hours after pollination is the best period for using pollen tube channel method and the female flowers like cavels at this time. In addition, stigma resection operation within the best operation period has on significant impact on conversion effectiveness.3、The2817bp promoter sequence of litchi TLP gene is cloned using PCR and sequence analysis identified abundant transcription regulation related cis-elements such as low temperature responsiveness element (LTR)、fungal elicitor responsiveness element (Box-W1)、meristematic tissue specific activated element (CCGTCC-box) and defense related element (TC-rich repeats).4. The CAMV35S promoter of pCAMBIA1304is replaced by four different length promoter of litchi TLP gene to construct plant expression vector (LcTLP-Q1-1304、LcTLP-Q2-1304. LcTLP-Q3-1304. LcTLP-Q4-1304).Leaf, pericarp, seeds and pulp are transformed by bombardment transformation respectively. The result of GUS histochemical staining shows that the promoter regulation of LcTLP is tissue-specific, with higher expression levels in leaf, pericarp, seeds and weak or barely detectable expression level in pulp.