| Tobacco?Nicotiana tabacum?is an important economic crop in China,cultivation and exploration of tobacco functions as the main industry of Yunnan Province.As a typical tropical-originated crop,the development and production of tobacco is quite sensitive to water demand.Drought and low temperature are important environmental factors limiting the yield and quality of tobacco,so the identification and selection of qualified germplasm with drought resistance of tobacco is a critical problem for agricultural production or breeding..At the present study,the method of membership function in fuzzy mathematics was used,to perform a comprehensive evaluation on drought/chilling resistance of six different tobacco cultivars,aiming at providing information on the selection and breeding for drought/chilling-resistant tobacco cultivars in the future.Jatropha curcas has attracted wide attention as a potential biofuel crop owing to its hardiness,high oil content,and suitability of seed oil for biofuel conversion.In particular,this small perennial tree or large shrub is well-adapted to extreme climates and arid/semi-arid regions,exhibiting the tolerance to these stress factors.Therefore,to elucidate the correlated physiological and molecular mechanism will provide detailed information on genetic breeding strategies aimed at improving traits with tolerance to various stress conditions.Taking use of Illumina Hi-seq 2000 technology,our previous investigations have isolated three stress-responsive candidates,JcP5CR?Jatropha curcas pyrroline-5-carboxylate reductase?,JcERF?Jatropha curcas ethylene response factor?and JcSP1?Jatropha curcas stable protein?,from J.curcas.Here,the complete coding region?CDS?of the candidate genes were amplified and subcloned into the binary expression vector for plant transformation,pBI121.Our data confirmed the overexpression of target genes conferred the tobacco seedlings various stresses tolerance to different extent,and will serve as a useful resource for genetic improvement of J.curcas in future.The main results are shown as follows: 1.Comparative screening and comprehensive evaluation of drought/chilling resistance of six tobacco varietiesTobacco seedling from six different varieties were subjected to air drought or 4 ? low temperature treatment respectively.Multiple biochemical and physiological index were determined,including electrical conductivity,water content of leaves,the content of malondialdehyde?MDA?,proline and soluble sugar.The method of membership function in fuzzy mathematics was used,to perform a comprehensive evaluation on drought/chilling resistance of six different tobacco cultivars The results showed that,Danyu NO.2 exhibited a relatively highest drought and chilling resistance,with due an average membership value at 7.48 and 0.72 respectively.YN116 and Yatas-6 were the quite opposite?1.62 for drought,0.263 for chilling?.2.Isolation of JcP5 CR gene from J.curcas,and stress tolerance analysis of transgenic tobacco plantsWe isolated the gene encoding P5CR?JcP5CR?from the leaves of Jatropha plants.The complete coding region?CDS?of JcP5 CR consisted of 828 nucleotides with an open reading frame encoding a polypeptide of 270 amino acid residue.Real time quantitative PCR analysis indicated that,when exposed to drought stress?10% PEG?for 48 h,accumulation of JcP5 CR transcrips in Jatropha leaves kept increasing,to approx.8.73-fold of that in untreated control.The proline content in wild type seedlings was approx.18.56 ug.g-1,accounting for 87% of that in transgenic plants.When exposed to air drought treatment for 3 d,proline content in leaves of transgenics increased to 9.14 mg.g-1?approx.1.33 times of wild type?.The germination assays of T1 generation seed showed that,when grown under chilling temperature?12 ??for 15 d,WT showed 70% germination whereas the transgenics showed 98.5 germination.At the presence of 200 mM NaCl for 15 d,germination percentage was reduced to 35.6% and 71.4% in WT and transgenics,respectively.The seed germination rate of wild type tobacco was only 35.6% after 15 days of PEG,while the germination rate of seed was 71.4%,exhibiting an increased tolerance to salinity and chilling stresses.3.Cloning of JcERF Gene of Jatropha curcas and Analysis of Tolerance of Transgenic TobaccoCloning get a ERF-like transcription factors in Jatropha curcas,its CDS region of 729 bp,coding protein of speculation conservative functional domains search,found with conservative AP2 / ERF domain structure.Functional element projections for its promoter regions,found ERF response element?ERE?in the transcription start site upstream1500 bp.Build dual plant expression vector pBI121-JcERF,and won the Overexpression of JcERF tobacco plants,and the tolerance of transgenic lines to high salt and low temperature stress was analyzed.Results showed that the The transgenic and wild tobacco leaves were placed in different concentrations of NaCl solution for analysis of the loss of green,found on the third day,about 30% of wild type blade has obvious albino,but transgenic tobacco leaves have not appeared albino,the performance of salt-resistant increase.After 24 h treatment with low temperature stress?0 ??,Transgenic tobacco electrolyte permeability is only wild-type tobacco 69%,While the content of proline and soluble sugar reaches at least wild type 3.82 times and 1.72 times.The germination rate of T1 seed tobacco at 12 ? was statistically analyzed,Three days later found the germination rate of genetically tobacco seeds is as high as wild type 3.89 times.These results indicate that the overexpression of the transcription factor JcERF entrusted a strong tolerance to transgenic tobacco on high and low temperature stress.4.Overexpression of JcSP1 gene resistance research in TobaccoThe transgenic tobacco seedlings with overexpressed JcSP1 were constructed,and subjected to stress-resistance analysis.When exposed to a high-temperature stress at 42 ? for 48 h,the relative electrical conductivity of transgenic tobacco leaves was only 87.56% of that from wild-type tobacco.Beyong that,the content of soluble sugar and soluble protein in transgenic tobacco leaves reached to approx.1.57 and 1.14 times of those in WT seedlings,exhibiting a relatively increased tolerance to high temperature stress. |
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