农杆菌介导的VgDGAT1a基因棉花茎尖转化体系优化研究

Improved Agrobacterium-mediated transformation protocol for VgDGAT1a gene based on shoot tip culture of cotton

为了将斑鸠菊(Vernonia galamensis)二酰甘油酰基转移酶基因(VgDGAT1a)导入棉花,创制转基因高油棉花种质,优化建立农杆菌介导的棉花茎尖转化体系,以‘中棉所49’茎尖为外植体,以HptⅡ基因为筛选标记,VgDGAT1a为目的基因,用农杆菌介导法,研究外植体培养时间、浸菌侵染时间、共培养时间及吸光度A600值等对棉花茎尖转化的影响。结果表明,在外植体培养3～5d、生长点纵切、菌液吸光度A600值0.6～0.9、浸菌40～60min、浸菌后暗培养1d和使用MSB+活性炭1g/L+头孢霉素400mg/L作为生根培养基的条件下能高效获得再生转化植株,从而为转基因高油棉花种质的创制提供了一种有效的方法。

Summary In past two decades, abundant researches have been published on transformation, regeneration, and genetic enhancement of cotton, especially Gossypium hirsuturn. Generally, genes conferring agronomic advantages have been introduced into plants via Agrobacteriurn-mediation or particle bombardment, and then the transgenic plants are regenerated through somatic embryogenesis from callus. However, the embryogenic callus-based regeneration is difficult and time-consuming in cotton. Therefore, it is necessary to establish an effective system for the Agrobacteriurn-mediated genetic transformation of shoot tip in cotton. Triacylglycerols （TAG） are a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bones. Diacylglycerol acyltransferase （DGAT; EC3.2.1.20） catalyzes the last step of TAG biosynthesis and it is the only key enzyme evolved in. DGAT1 and DGAT2, as two types of DGAT in eukaryotes, belong to different gene families. And previous studies have reported that the expression of DGAT1 increased seed oil content and mass. In order to get new cotton germplasm with high oil content, we used the shoot tips of ＇ Zhongmiansuo 49＇ as explants and introduced an improved vector carrying a selection marker Hpt Ⅱ gene and a target VgDGATla gene into cotton via Agrobacterium-mediated transformation. This improved Agrobacterium-mediated transformation and regeneration system were established by optimizing different parameters such as pre-culture period of seeds, concentration of Agrobacterium in solution, immersing time, co-cultivation period and components of MSB [Murashige and Skoog （MS） medium - vitamins of Gamborg＇s （B5） medium3. Cotton seeds （Gossypium hirsutum L. cv. ＇Zhongmiansuo 49＇） were decorticated manually and surface sterilized in 0.1 HgC1 for 8 min, followed by rinsing with sterile distilled water for five times. The cotyledons were removed from 3-day-old to 5-day-old in vitro germinated seedlings and the shoot tips were cut in lengthwise to decrease the damage of meristematic cells. The strain EHA105 was grown overnight on a shaker at 200 r/min and at 28 ℃ until the A600 value of bacterial concentration reached 0.6 - 0.9. The suspension cells were centrifuged at 5 000 r/min for 10 min and the pellets were resuspended in an equivalent volume of liquid coultivation medium [MSB ＋ 200 μmol/L acetosyringone （AS）3. The treated explants were immediately immersed into prepared Agrobacterium suspension containing pCAMBIA1301, a binary vector carrying the VgDGATla gene, for 40 -60 minutes. The tips were blotted dry on sterile filter paper and transferred into the co-cultivation solid medium at 28 μ in dark. After co-cultivation for 1 day, shoot tips were transferred into root induction medium containing MSB, 1 g/L activated charcoal, 400 mg/L cephalothin （Cef） and solidified with 0.2% （W/V） phytagel, then grew in a growth chamber with stringent light, temperature and humidity control. In the following 3 - 4 weeks, 5-leaf-plants were transferred to plastic pots containing soil matrixes （1 ： 1 of turf and vermiculite）. Besides the optimized genetic transformation protocol of cotton shoot tip as stated above, it is found that 50 mg/L hygromycin （Hyg） could accurately distinguish the resistant plants. In addition, reduplicated selections improved accuracy. In conclusion, this study describes an optimized transformation protocol for shoot tip of ＇ Zhongmiansuo 49＇ with an A. tumefaciens strain EHA105 harboring DGAT1 gene, and proves that it is an efficient and economical method to obtain transgenic plants based on the results of different and important parameters influencing the transformation efficiency.