甘蔗热带种金属硫蛋白家族基因的克隆及响应重金属胁迫的表达分析     

高世武  傅志伟  陈云  林兆里  许莉萍  郭晋隆 《作物学报》2020,46(2):166-178

金属硫蛋白是一类富含巯基的低分子量蛋白,在植物的重金属解毒及细胞氧化还原调控等方面起重要的作用。本研究以甘蔗热带种Badila组培苗为材料,分别测定了其在CdCl2、ZnSO4和CuCl2水溶液培养条件下地上部和地下部的重金属含量,结果显示其对上述3种重金属有较强的耐受与富集能力。继而克隆了ScMT1(登录号为KJ504373)、ScMT2-1-5(登录号为MH191346)和ScMT3(登录号为KJ5043704)3个金属硫蛋白家族基因,它们分别属于植物MT亚家族中的MT1、MT2和MT3型基因。ScMT1含有1个内含子和2个外显子,开放阅读框(Open Reading Frame,ORF)长228 bp,编码75个氨基酸;ScMT2-1-5含有2个内含子和3个外显子,ORF长246 bp,编码81个氨基酸;ScMT3含有1个内含子和2个外显子,ORF长198 bp,编码65个氨基酸。RT-qPCR显示,Cd2+胁迫下,在甘蔗地上部和地下部,ScMT2-1-5均连续显著上调表达,而ScMT1的上调应答出现延迟。ScMT3在地上部的上调应答出现延迟,在地下部呈“扬-抑”趋势,提示甘蔗响应Cd^2+胁迫过程中ScMT2-1-5起更积极的作用,ScMT1参与胁迫后期的分子响应,而ScMT3不起主导作用。Cu^2+胁迫下,地上部ScMT1连续显著上调表达,ScMT2-1-5和ScMT3呈总体上调的表达趋势;地下部,ScMT1和ScMT2-1-5的上调表答均出现延迟,仅在胁迫后期显著上调表达,而ScMT3仅在胁迫前期显著上调表达。该结果提示了ScMT1、ScMT2-1-5和ScMT3在Cu2+胁迫响应过程中的协作关系,三者共同参与了地上部的胁迫响应,其中ScMT1起更积极的作用;此外三者还先后参与了地下部对Cu^2+胁迫的分子响应。Zn^2+胁迫下,ScMT1和ScMT3分别仅在地上部和地下部显著上调表达;ScMT2-1-5在地上部和地下部均呈“扬-抑”的应答趋势;提示了在甘蔗响应Cd^2+胁迫应答过程中ScMT1和ScMT3分别在地上部和地下部起主要作用,ScMT2-1-5参与了胁迫前期的分子响应。ScMT1、ScMT2-1-5和ScMT3在甘蔗不同组织中及在重金属(Cd^2+、Zn^2+或Cu^2+)不同累积水平下呈现出相似或互补的应答特性,提示上述甘蔗MT家族不同成员在重金属解毒及细胞氧化还原调控等方面产生了功能分化,且三者在应对过量Cd^2+、Zn^2+或Cu^2+对甘蔗组织造成伤害的过程中存在时空上的协同作用。该研究为深入理解多倍体植物甘蔗中MT家族各成员基因在重金属耐受过程中的协同作用机制奠定了基础。  相似文献   

Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation     

Thi Thanh Nhan Pham  Thi Ngoc Lan Nguyen  Thi Ha Bui  Huu Quan Nguyen  Thi Tam Nguyen  Van Son Le 《The Journal of Horticultural Science and Biotechnology》2019,94(5):591-598

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter  相似文献   

转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测     

冯翠莲  万玥  冯小艳  王俊刚  赵婷婷  王文治  沈林波  张树珍 《热带作物学报》2021,42(9):2468-2477

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。  相似文献   

Changes in photosynthesis,growth, and sugar content of commercial sugarcane cultivars and Erianthus under flood conditions     

Thanankorn Jaiphong  Jun Tominaga  Kenta Watanabe  Ryuichi Suwa  Masami Ueno 《Plant Production Science》2017,20(1):126-135

Sugarcane (Saccharum spp.) is an economical crop in the tropical and subtropical countries. However, because of global climate change, flooding has become problematic, particularly during the rainy season, in Thailand. We investigated the effects of floods on three commercial sugarcane cultivars, namely NiF8, U-thong 6 (UT6), and U-thong 9 (UT9), as well as Erianthus spp. Growth was assessed using a pot experiment in a glasshouse with two treatments: (1) control and (2) 60 d of flooding followed by 30 d of normal conditions. In comparison with control, during prolonged flooding, Erianthus showed greatly decreased CO₂ assimilation, whereas NiF8, UT6, and UT9 showed slightly declined CO₂ assimilation. Growth in plants subjected to 60 d of flooding was less influenced by floods while sucrose content was not affected except in UT6. During flooding, some roots died, resulting in plants compensating adventitious roots to offset the negative effects of root death and to assist them in maintaining their growth, which appeared from the submerged nodes, with different characteristics for each cultivar. However, 30 d after draining, roots remained damaged, while adventitious roots died, resulting in lesser growth as compared with the control, but it did not significantly affect sucrose content and sugar yield. This study suggests that sugarcane plants need to produce the adventitious roots to compensate their roots’ death during flooding and require time to recover their root system after flooding for obtaining the optimum yield and quality at harvest.  相似文献   

甘蔗叶不同还田方式对土壤养分的影响   总被引：1，自引：0，他引：1  

文少白  葛畅  李明  邹雨坤  侯宪文 《热带作物学报》2017,(8):1423-1426

为减轻秸秆焚烧所带来的环境问题和充分利用甘蔗叶资源,在大田环境下动态监测甘蔗叶粉碎、焚烧和深埋3种还田方式对土壤养分的影响。结果表明:甘蔗叶粉碎还田和深埋还田有利于甘蔗叶中有机碳及氮、磷、钾养分的缓慢释放;甘蔗叶焚烧还田提高了土壤碱解氮含量5.6~20.9 mg/kg和速效磷含量0.8~4.0 mg/kg,促进土壤中氮磷向有效态转化,焚烧还田初期显著增加了土壤速效钾的含量12.3~18.3 mg/kg,但焚烧会损失部分甘蔗叶中碳氮元素,也造成环境污染。综合分析认为甘蔗叶粉碎还田是比较省工、方便的处理方式。  相似文献   

Composition of organic C fractions in soils of different texture affected by sugarcane monoculture     

Anna Kusumawati  Benito Heru Purwanto  Makruf Nurudin 《Soil Science and Plant Nutrition》2020,66(1):206-213

ABSTRACT

Sugarcane is a strategic commodity in Indonesia. It is usually raised in a monoculture system. There is a lack of information about the effects of extended sugarcane monoculture on the soil carbon fraction. The aim of this study was to determine the relative changes in the soil organic C fractions in response to the duration of sugarcane monoculture on Entisols, Inceptisols, and Vertisols. The measured variables were the percentages of sand, silt, and clay, organic matter (OM), total nitrogen (TN), pH (H₂O), cation exchange capacity (CEC), NH₄ ⁺, NO₃ ^-, labile carbon fraction (soil carbon mineralization (C-Min), soil microbial carbon (C-Mic), and carbon particulate organic matter (C-POM)), and stable carbon fraction (humic and fulvic acids). Soil type with sugarcane monoculture period had significant influences on the percentages of clay, sand, silt, CEC, and pH (H₂O). Soil type and sugarcane monoculture period had no apparent significant effect on C-Min or C-POM but did significantly influence C-Mic. The humic and fulvic acid levels in all three soil types were affected by the duration of sugarcane monoculture. To establish the impact of long-term sugarcane monoculture on the physicochemical properties of soils with various textures, it is more appropriate to measure the soil stable carbon fractions such as humic and fulvic acid rather than the soil labile carbon fractions such as C-Min, C-POM, or C-Mic.  相似文献   

水稻OsUBA基因的表达及其在促进种子萌发和开花中的功能     

张双双  王立伟  姚楠  郭光艳  夏玉凤  秘彩莉 《作物学报》2019,45(9):1327-1337

自噬是将功能异常或不需要的胞内组分降解的细胞学过程,广泛参与真核生物的生长发育过程、对营养缺乏的响应及生物/非生物胁迫反应。NBR1 (Next to BRCA1 gene 1, NBR1)是在植物中发现的最重要的自噬受体,但有关植物NBR1类自噬受体的研究较少,水稻中此类蛋白的研究还是空白。本文通过RT-PCR方法,从水稻日本晴幼苗的cDNA中克隆到一个含有泛素相关结构域(Ubiquitinassociated,UBA)的基因,将其命名为OsUBA。OsUBA的开放阅读框长2538 bp,编码845个氨基酸残基。OsUBA属于水稻中的NBR1类蛋白。OsUBA的启动子区有多个与光、逆境胁迫及激素反应相关的元件; OsUBA基因在水稻花药、正在萌发的种子以及根中的表达量较高,在茎和叶中也有表达; 200μmol L~(–1) ABA处理显著抑制OsUBA的表达,100μmol L~(–1) GA处理后OsUBA的表达略有升高。对OsUBA过表达水稻株系的研究表明,转基因水稻种子的萌发比野生型更快, ABA (3μmol L~(–1))处理显著抑制OsUBA过表达水稻株系种子的萌发, GA (100μmol L~(–1))处理对OsUBA过表达水稻株系种子的萌发略有促进;OsUBA过表达水稻株系的开花时间较野生型明显提前。这些结果表明,水稻NBR1蛋白基因OsUBA的表达和功能可能与对开花时间和种子萌发的调控以及生物/非生物胁迫反应有关。  相似文献   

花粉管通道法转基因抗虫棉外源基因的整合方式   总被引：4，自引：0，他引：4  

张保龙  倪万潮  张天真  郭旺珍  徐英俊  姚姝 《江苏农业学报》2004,20(3):144-148

对花粉管通道法培育的转Bt基因抗虫棉GK19、GK22、1016、1018、1022、1025、GK5、GKsu12和SGK1进行Southern杂交分析。结果表明,目的基因能够完整整合到基因组里,HindⅢ酶切位点有可能发生甲基化或丢失。除1018以外的上述8个抗虫棉的分析结果还表明,载体骨架没有整合到棉花基因组中。  相似文献   

转基因动物研究进展   总被引：4，自引：0，他引：4  

胡建宏李青旺  王立强江中良于永生  解广勤 《家畜生态》2004,25(2):50-53

转基因动物是现代生物技术中一个极其重要的研究领域，目前已经有转基因小鼠、兔、绵羊、山羊、猪、牛、鸡和鱼等多种转基因动物问世。本文综述了转基因动物的制作方法、转基因动物的应用研究以及所取得的重要成就，并指出了转基因动物存在的主要问题，展望了其发展前景。  相似文献   

人血栓调节蛋白基因的表达及转基因小鼠的建立     

陈红英  苟克勉  王晓琳  陈学进  黄秀英  孙方臻 《中国兽医学报》2003,23(2):115-118

用PCR方法从人的基因组DNA中扩增了人血栓调节蛋白（hTM)基因，将其克隆到带有人EF－1α启动子的pEF-neo哺乳动物表达载体上，得到表达质粒pEF-TM。在转染pEF-TM的COS－1细胞中，hTM得到了瞬时表达，并且正确地定位到细胞膜上。用pEF-TM转染猪内皮细胞（PEC），经G418筛先得到稳定转染的克隆。凝血活性测定结果显示，表达hTM的PEC凝血时间明显延长，表明其抗凝血能力增强。通过显微注射方法，得到了1只F0代hTM转基因小鼠。Southern杂交结果表明，该转基因小鼠整合了9个拷贝的pEF-TM DNA,并能将外源DNA遗传给后代。  相似文献