水稻遗传转化方法研究与应用进展

目的建立以日本晴愈伤组织为受体、草甘膦抗性基因CP4为选择标记基因的高效水稻遗传转化体系。方法以粳稻日本晴的成熟胚为试验材料,通过农杆菌介导法,将含有草甘膦抗性基因CP4的P1300载体转化日本晴的愈伤组织,分别用质量浓度为10、20和40 mg/L的草甘膦进行3轮抗性筛选,诱导分化和再生成苗;利用1000 mg/L草甘膦对移栽成活的阳性苗进行抗性鉴定。结果获得145株转基因幼苗,PCR检测阳性株为128株,阳性率达88.27%;经过3轮抗性筛选后,转基因幼苗的阳性率提高;移栽成活的99株阳性苗中,65株表现为抗性,抗性率为65.66%。结论成功建立了以草甘膦为筛选标记的水稻转基因体系,该体系为水稻育种提供了材料,并对以抗草甘膦基因作为筛选标记的水稻转基因和一些基因功能验证提供了技术支持。     

Effect of Phytosulfokine-a on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine- α （PSK- α ）, a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D.     

Constructing the barley model for genetic transformation in Triticeae

Barley(Hordeum vulgare L.) is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.     

Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon （Citrus Limon （L.） Burm. F. cv. Eureka）

The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.     

农杆菌介导法向小麦茎尖导入DREB1A基因的研究初报

为了研究以小麦茎尖为受体进行农杆菌转化的可行性,选用小麦品种H6756,以茎尖为受体进行了农杆菌转化.构建了含有拟南芥逆境诱导转录因子DREB1A及除草剂bar基因的表达载体pC3300IS-DREB1A,酶切鉴定此载体,结果证明其连接完全正确,具有转录和表达的功能.农杆菌介导法向小麦茎尖导入DREB1A基因,共获得247株转化苗.转化苗经除草剂筛选,获得66株抗性苗,对抗性苗进一步进行PCR检测,有30株抗性苗扩增出特异性片段,转化率达到12.1%,初步说明本试验中以小麦茎尖为受体的转化系统用于基因转化是可行、高效的.     

Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine(PSK)is a new peptide plant hormone,which was isolated in the conditioned medium of cultures derived from both monoco-tyledonous and dicotyledonous plants,such as Asparagus officinalis mesophyll[1],rice[2],zinnia[3],and carrot[4].PSK has two types of structure:PSK-αand PSK-β.The former is a sulfated pentapeptide[H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH],the latter is a sulfated terapeptide[H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH].Both are heat-stable,susceptible to pronase…     

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

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     

农杆菌介导的油菜遗传转化研究进展

综述油菜遗传转化中再生体系的建立、农杆菌侵染方法、适宜菌株及载体选择等方面的研究进展,并对转化中寄主基因型、目的基因及其启动子的选择、筛选方法、外源基因稳定性等方面存在的问题进行了分析与讨论,为优化油菜等芸薹属的遗传转化提出可行性建议.     

影响农杆菌介导的大豆基因转化因素的研究

通过农杆菌介导法用含NPT－Ⅱ和Barnase基因导入到大豆幼胚，大豆子叶节叶，然后经过含Kan的筛选培养基中连续筛选，获得一批Barnase转基因植株。经PCR扩增结果，证实此基因已整合到大豆中，在当代植株中生物学特性观察，花粉粒不育率在98％。     

根癌农杆菌介导ACS反义基因转化香蕉的研究

以香蕉(Musa spp.)品种‘天宝蕉’(Musa spp.,AAA类群)为试材,对以根癌农杆菌介导法的香蕉遗传转化体系进行较全面的研究,并以该系统进行了ACS反义基因转化香蕉的研究。结果表明:不经预培养的香蕉茎尖横切薄片,侵染前用附加0.1 mg/L甘露醇的高渗固体培养基前处理4 h,农杆菌重悬液浓度为OD600在1.0左右,重悬液中含100 g/L蔗糖,接菌时间为10~15 min,于26℃黑暗条件下共培养4 d,共培养培养基pH值为5.8是较为适合的转化条件;采用附加100 mg/L卡那霉素、2 mg/L AgNO3筛选培养基对共培养后的香蕉横切薄片进行筛选,共获得5个转ACS反义基因的抗性芽系;经GUS组织化学法及PCR检测,gus基因已整合进香蕉基因组。