烟草抗非生物逆境分子育种研究进展

双子叶模式植物烟草是重要的经济作物,烟草抗非生物逆境分子生物学的研究一方面可以验证异源抗逆境基因的功能,另一方面有可能获得抗非生物逆境能力提高的烟草新品种。综述了近年来国内外利用转基因技术,在验证抗非生物逆境基因功能、提高烟草抗寒、抗旱、抗盐和重金属等非生物逆境方面的研究进展,以期为其它作物的抗逆研究提供参考。     

UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控

用根癌农杆菌介导法将源于紫穗槐的尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）基因、反义4-香豆酸辅酶A连接酶（4CL）基因以及两者的双价基因分别转移至烟草中。PCR和Southern杂交检测证实外源基因已整合到转基因烟草基因组中。测定全纤维素和Klason木质素含量的结果显示,增强UGPase基因的表达可提高转基因植株的纤维素含量,但对木质素含量没有影响;抑制4CL基因的表达可显著降低转基因植株的木质素含量,但对纤维素含量没有影响;转移双价基因的转基因植株中纤维素含量增加而木质素含量降低。     

我国烟田昆虫群落的划分及其特征分析

根据纬度、海拔、温度、降雨、日照等地理、气象因子指标,对我国71个植烟市和(或)县及生态条件独特的市和(或)县进行了聚类分析,聚类结果与我国烟草的7个一级种植区基本吻合,我国烟田烟草昆虫群落可依此划分为东北、黄淮、长江中上游、长江中下游、西南、南部和北部西部等7个。各群落的重要烟草昆虫种类有所不同,烟草昆虫天敌的种类差别更大。其中西南群落食物网最复杂。     

嗜水气单胞菌HBNUAh01 外膜蛋白A 基因在烟草叶片细胞中的瞬时表达

【目的】从嗜水气单胞菌(Aeromonas hydrophila)HBNUAh01中克隆外膜蛋白A(outer membrane proteinA,ompA)基因并在烟草(Nicotiana tabacum)叶片细胞中瞬时表达该蛋白。【方法】以嗜水气单胞菌HBNUAh01为模板进行嗜水气单胞菌外膜蛋白A(AhompA)基因片段的PCR扩增,并将其克隆到pEASY-Blunt Simple载体中以进行测序。测序正确的AhompA基因序列与含有黄色荧光蛋白(yellow fluorescentprotein,YFP)基因的表达载体pCAMBIA1300构建重组表达载体。将该重组表达载体转化到农杆菌(Agrobacterium tumefaciens)GV3101感受态细胞中,随后用阳性转化子转染烟草叶片细胞。使用激光扫描共聚焦成像系统(Confocal Laser Scanning Microscope)检测观察融合表达AhompA基因的黄色荧光蛋白并采用RT-PCR检测AhompA基因在烟草叶片中的转录情况。【结果】从嗜水气单胞菌HBNUAh01中克隆出大小为1032 bp的AhompA基因序列,并在烟草叶片中成功表达AhompA和YFP的融合蛋白。【结论】AhompA基因在烟草叶片细胞中的成功表达为进一步研究利用植物疫苗防治嗜水气单胞菌引起的水产动物疾病奠定了基础。     

烟草种间杂交亲和性

以野生烟草Nicotiana alata、N.rustica、N.repanda、N.stocktonnii与栽培烟草K326、红花大金元、Yun87、Yun97为材料,进行种间正反杂交,研究种间杂交亲和性。田间观察杂交后的坐果情况并统计坐果率,采用显微荧光染色观察授粉后花粉管在雌蕊上的生长情况,并结合杂交后代萌发检测的方法。结果表明：N.rustica、N.repanda、N.stocktonnii与栽培烟草杂交不亲和。N.rustica花粉能够穿过K326花柱,N.repanda和N.stocktonnii花粉在K326柱头上很少萌发生长。N.alata花粉可以穿过K326的花柱,并得到果实,但是萌发实验显示其种子无活力。N.alata作为母本与栽培烟草杂交不亲和。     

转天山雪莲sikPIP3基因烟草的获得及抗逆性鉴定

利用PCR技术从实验室建立的天山雪莲DNA文库中克隆了天山雪莲质膜水孔蛋白基因sikPIP3,构建了植物表达载体pBI121-sikPIP3,通过农杆菌介导法转化烟草品种NC89,经PCR和RT-PCR检测证明目的基因成功导入并得到了表达,采用水分胁迫进行抗旱分析和采用冷冻胁迫进行抗寒性分析。结果显示:(1)克隆出具有水孔蛋白基因特性的sikPIP3基因。(2)经断水干旱处理,转基因烟草的生长表型优于野生型烟草,特别是在断水9d的情况下,野生型烟草已经完全萎蔫,转基因烟草萎蔫症状较轻;生理指标测量结果显示,转基因烟草的相对电导率和MDA的含量低于野生型烟草,相对含水量和CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗旱性高于野生型烟草。(3)经不同温度胁迫处理,转基因烟草的生长表型优于野生型烟草,特别是在-4℃冷处理6h情况下,野生型烟草已经完全萎蔫,转基因烟草只出现少量伤斑;生理指标分析结果表明,转基因烟草的相对电导率和MDA的含量低于野生型烟草,CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗寒性高于野生型烟草。综合结果表明:sikPIP3基因在抗逆基因工程方面具有较高的应用前景。     

ICTV第九次报告对病毒分类系统的一些修改

正国际病毒分类委员会(International Committeeon Taxonomy of Viruses,ICTV)于2012年发表了最新的病毒分类第九次报告[1]。在这个长达1327页的报告中,将目前ICTV所承认的2284种病毒和类病毒归入349个属、19个亚科、87个科和6个目,     

3种渗透剂对转基因烟草根系枯草芽孢杆菌纤溶酶(BSFE)分泌的调节

用0.05%～8.00%的甘露醇、山梨醇和聚乙二醇6000等3种渗透调节剂可提高转枯草芽孢杆菌纤溶酶(Bacillus subtilis fibrinolytic enzyme, BSFE)转基因烟草(Nicotiana tabacum L.)根系BSFE的分泌表达水平,其水培液BSFE活性在15 d内基本呈抛物线型变化趋势.经3种渗透剂处理后转BSFE基因烟草水培液的BSFE活性峰值明显高于对照,且出现时间比对照相对延迟1-2d.甘露醇、山梨醇和聚乙二醇6000可作为该转基因烟草根系BSFE分泌表达的有效化学调节剂.     

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifolafrondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126-2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bifunctional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase(TPP) of E. coil, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene.It has been reported that transgenic plants with E. coil TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S-35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.     

Isolation and Expression Pattern Analysis of Two Ferritin Genes in Tobacco

For understanding of the ferritin gene expression pattern and the mechanism of iron homeostasis in tobacco (Nicotiana tabaccum L.) plants, two full-length ferritin cDNAs, NtFerl and NtFer2, were isolated from tobacco seedlings and characterized. These cDNAs are 1 214 and 1 125 bp nucleotides and encode 251 and 259 amino acid residues, respectively. The deduced amino acid sequences showed that two tobacco ferritins share the same characteristics as the plant ferritins from Arabidopsis, soybean, and maize. Southern blotting analysis indicated that both NtFerl and NtFer2 were probably multicopy genes in the tobacco genome. Northern blotting analysis indicated that iron loading of tobacco plantlets increased the ferritin mRNA abundance and that NtFerl expression was higher and more sensitive to iron than NtFer2 expression. Furthermore, NtFerl was expressed in both leaves and roots, whereas NtFer2 was expressed mainly in leaves.