| Rice is one of the most important food crops as well as a model plant formonocotyledon research. With the development of rice genome project, it becomesincreasingly a hotspot to analyze the genes function and to apply them in geneticengineering research. Environmental stresses such as cold, high salinity and drought aremajor obstacles affecting plant growth and crop productivity. Our research focused onidentifying the TFIIIA type zinc finger protein genes involved in abiotic stress, andexpecting to provide new targets for producing tolerance-enhanced transgenics. Noveltiesachieved in this study are as follows:Two novel TFIIIA-type zinc finger protein genes RZF5 and RZF71 was identifiedfrom rice (Oryza sativa L. subs. japonica) by bioinformatics method and RT-PCR approach.Sequence analysis showed that RZF5 and RZF71 both contained two typical C2H2 zincfinger domains. RZF5 encodes a 17.95 kDa polypeptide with 171 amino acids and RZF71encodes a 25 kDa polypeptide with 250 amino acids. RT-PCR analysis showed that RZF5and RZF71 were constitutively expressed in various rice tissues including root, stem, leaveand spike, and strongly induced by 150 mmol·L-1NaCl and 20％PEG6000. But both geneswere not induced by low temperature (4℃) and 0.1 mmol·L-1 ABA treatments.Data analysis of rice genome shows that RZF5 is located on the long arm ofchromosome 1 and RZF71 is located on long arm of chromosome 12. About 1500bpsequences of the upstreams form the translation start codons of RZF5 gene and RZF71 genewere analyzed by MapInspector program, respectively. The results show that there are nineputative cis-acting elements related to abiotic stress in RZF5 promoter regions, includingone ABRE (abscisic acid response element), two CRT/DREs(C-repeat/dehydration-response elements), five MRSs (sequence homologous to MYBrecognition sites) and one W-box were found within RZF5 promoter, and there were sixelements in RZF71 including one CRT/DRE, three MRSs, one ICE (inducer of CBFexpression) element and one W-box.About 1500 bp sequence in the promoter region of the RZF5 gene was fused to GUSreporter gene in pCAMBIA1301 vector and then was transformed into rice callus by Agrobacterium-mediated transformation. Histochemical analysis revealed, that the GUSactivity was induced both in the 150 mmol·L-1 NaCl and 20％PEG6000 treatments, but notin the cold (4℃) treatment. To investigate the subcellular localizations of RZF5 and RZF71protein, RZF5 and RZF71 gene were fused in the green fluorescence protein (GFP) reportergene, respectively. These fusion genes were introduced to onion epidermal cells and neecallus cells respectively. The results showed that the fusion proteins were localized in thenucleus, respectively, while the control was distributed throughout the cells.To find out the functions of these two zinc finger proteins, we generated RZF5-sensetransgenic tobacco plants by Agrobacterium-rnediated transformation. Among eighteenplants of the T0 generation, the growth of sixteen plants was obviously suppressedcompared to the non-transgenic ones. And the transgenic plants of the T1 generation wereanalyzed for abiotic stresses. The measurements showed that the relative root length of thetransgenic seedlings were elongated 36.1％and 17.4％compared to the normal one under100 mmol·L-1 and 200 mmol·L-1 NaCl treatments, respectively, whereas the non-transgenicones could only elongated 15.3％and 1.4％, respectively. Under 300 mmol·L-1 and 500mmol·L-1 Mannitol stress, the relative root length of the transgenic seedlings elongated98.0％and 67.6％, respectively, whereas the non-transgenic ones could only elongated62.2％and 24.3％. There was no significant difference between the transgenic seedlings andthe non-transgenic ones under controlled cold stress. Thus, the overexpression of RZF5 inthe transgenic tobacoo plants confered tolerance to high salt and osmotic stress.Transgenic RZF5 (sense, antisense, hairpin) rice plants of were generated byAgrobacterium-mediated transformation. The growth of RZF5-sense transgenic plants wasretarded. Average plant height of sixteen T0 generation of RZF5-sense transgenic plantswere 40.9％to 57.1％, to that of non-transgenic plants. But transgenlc RZF71 (sense,antisense, hairpin) plants exhibited neither growth inhibition nor visible phenotypicalterations. Transgenic lines were analyzed for cold and NaCl stress in the T1 generation.Electrolyte leakage rate and plant morphology were compared between transgenic lines anduntransformed after clod stress 36 h. The results showed the growth of three independentRZF5-sense lines were no visible cold symptom and electrolyte leakage rate (8.54％,11.20％and 1.70％) lower than those of untransformed (84.18％). Data collected forpercentage of germination showed three independent transgenic RZF5-sense lines weremore tolerant than untransformed and germination rate of three transgenic lines were79.5％, 82.5％and 92.5％, whereas untransformed were only 62.5％under 150 mmol·L-1 NaCl stress. Thus overexpression of RZF5 conferred tolerance to cold and salt stress whiletransgenic RZF71 rice plants did not. These results suggest RZF5 may be quite useful inenhancing plant tolerance to abiotic stress.Anther culture has become a powerful method to research plant physiology,biochemistry and genetics. And it has been playing important roles to research genesregulation and expression mechanism by anther culture in physiology and molecularbiology. Doubled haploids population based on anther culture is perfect material formolecular marker assistant breeding and genetic mapping. It is a topic that how to improveanther culture efficiency for scientists. In this reseach, the anthers of F1 progenies fromthree crosses between japonica and indica rice varieties and their parents, and one japonicavariety Wuyunjing 8 were used to induce the calli in the M8 medium supplemented withdifferent concentrations and combinations of plant hormones and organic nutriments. Ahighly efficient system of anther culture was established for F1 progenies between japonicaand indica rice varieties. The results showed that the highest rate of callus induction(4.1％-4.5在for japonica/indica F1 hybrid ) were obtained in the M8 medium supplementedwith 2,4-D (2 mg.L-1), NAA (1mg.L-1) and KT (0.2 mg.L-1). The induction rate could befurther increased up to 20.5％-27.3％in this medium added with hydrolyzed casein andyeast extract; It could be obtained, the 14.3％-25.0％of plant regeneration rate(japonica/indica F1) in the M8 medium added with 6-BA (2.5 mg'L-1) and KT (0.5mg.L-1). |
PDF Full Text Request