Vector Construction And Genetic Transformation Toward Specific Expression Of Resistant Genes In Peanut

Peanuts is vulnerable to Aspergillus flavus infection, leading to aflatoxins contamination, which is a serious problem of food security in peanut. Peanut pod shell and seed coat play a key role against Aspergillus flavus invasion. Therefore, changing chemical composition and structure characteristics of carp and seed coat of peanut will breed resistant varieties. So far, however, conventional breeding method do have bred some resistance varieties with unstable performance in field and takes longer time for breeding. To deal the problem, our laboratory was the first time to propose and to carry out genetic engineering toward breeding high resistant varieties. In order to meet the security requirements, we have been cloning specific promoters for driving resistance genes to express specifically in carp and seed testa and also developed marker-free transgenic technology. In the paper, available tissue-specific promoters are employed to construct carp or seed testa-specific expression vectors; tool vector with characteristic of dex-induced removement of antibiotics is used to accommodate bivalent resistant genes; and previously constructed vectors are taken to study genetic transformation techniques. The results are as follows.1. Six plant expression vectors for enhancing the resistence or changing the chemical component or composition structure of peanut carp or seed coat were constructed with carp-specific expression promotors (8# and 13#) and four exogenous genes (CHI, GLU, COMT, RS).2. Using single valent vectors, harboring carp/seed coat-specific promoters (8 #, 13 # and S19 #) drivng CHI, GLU, CBF2, RS genes, respectively, and the vector with characteristic of removing antibiotic genes upon induction of Dex, two plant bivalent expression vectors were constructed, which are named as pLoxp-8-CBF2-S19-RS and pLoxp-8-CHI-13-GLU.3. Regeneration system and genetic transformation techniques with hypocotyle axises as explant are optimized. The highest rate of bud induction will produce in shoot induction medium formula of MMS +30 mg/ L BA +0.5 mg/L 2.4-D; maximum elongation will be when adopting elongation medium MMS +3 mg/L BA +1 mg/L NAA +2g/L active carbon; by successive screening, the concentration of hygromycin is 10 mg/L in the medium for the first two weeks and then 20 mg/L for two or three weeks following it, which will appear the best effect of selection; the concentration of hygromycin is 1 mg/L in rooting culture stage for the best selection, growing in medium for 14 to 28 days, when root elongation to 3-4 cm, the graft should be performed.4. Employing optimized transformation system, the above constructed vector and previously constructed ones were transformed, we obtained T0 trangenic plants with CHI and GLU genes.Through the above studies, we constructed some carp/testa-specific expression vectors, two induced-removing antibiotics plant bivalent expression vectors, and optimized peanut transgenic regeneration and screening system, which will lay basis for safely solving peanut aflatoxin contamination by gene engineering.