發(fā)布時(shí)間：2018-01-19 01:13

  本文關(guān)鍵詞： 棉花 草甘膦 EPSPS-G6 農(nóng)桿菌活體轉(zhuǎn)化方法 草甘膦抗性 產(chǎn)量 纖維品質(zhì)　出處：《浙江大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：棉花是唯一可由種子生產(chǎn)纖維的農(nóng)作物,棉花生產(chǎn)需要投入較多勞動(dòng)力。由于勞動(dòng)力的轉(zhuǎn)移,我國的人工價(jià)格不斷上漲,植棉的比較效益和經(jīng)濟(jì)效益下降,導(dǎo)致近年我國棉花生產(chǎn)的連年滑坡。我國的棉花生產(chǎn)仍以人工作業(yè)為主,其中棉田除草是植棉用工的主要內(nèi)容之一,占植棉用工的1/3以上。草甘膦(Glyphosate)是一種典型的廣譜性、內(nèi)吸傳導(dǎo)型滅生性除草劑,是雜草控制的首選除草劑。本研究將具有自主知識產(chǎn)權(quán)的抗草甘膦除草劑基因(EPSPS-G6)構(gòu)建進(jìn)植物表達(dá)載體,用農(nóng)桿菌活體轉(zhuǎn)化方法將目的基因轉(zhuǎn)入陸地棉推廣品種中棉所49,創(chuàng)制高抗草甘膦棉花種質(zhì)系,分析其抗性表現(xiàn)及相關(guān)農(nóng)藝性狀,探討其生產(chǎn)應(yīng)用前景。主要研究結(jié)果如下：1、利用本實(shí)驗(yàn)室構(gòu)建的表達(dá)載體pCAMBIA1300-EPSPS-G6,酶切獲得目的基因EPSPS-G6.并與空載體pCAMBIA2301連接構(gòu)建棉花高效表達(dá)載體——pCAMBIA2301-EPSPS-G6,去除原載體中的NPII篩選標(biāo)記基因,將目的基因作為篩選標(biāo)記。2、對本實(shí)驗(yàn)室創(chuàng)制的農(nóng)桿菌活體轉(zhuǎn)化方法進(jìn)行優(yōu)化。結(jié)果表明,含50mg/L乙酰丁香酮(acetosyringone,AS)的轉(zhuǎn)化液可將陽性率提高至22.18%,是對照(9.72%)的2.28倍。此外,不同轉(zhuǎn)化時(shí)間轉(zhuǎn)化效率差異顯著,其中開花當(dāng)天中午12：00～14：00轉(zhuǎn)化得到陽性率最高(22.18%),下午13：00～15：00次之(18.86%),上午8：00～10：00轉(zhuǎn)化效率最低(11.29%)。3、采用優(yōu)化的農(nóng)桿菌活體轉(zhuǎn)化方法,將EPsPS-G6基因?qū)氲疥懙孛尥茝V品種——中棉所49,共收獲轉(zhuǎn)化種子14.3 kg,T0轉(zhuǎn)化植株經(jīng)20 mM草甘膦噴施,陽性率達(dá)22.18%。經(jīng)PCR驗(yàn)證,共獲得了35株含EPSPS-G6基且抗草甘膦的To轉(zhuǎn)基因植株。4、T1轉(zhuǎn)基因后代的抗性分離比例從1/4-3/4不等。對T1陽性植株進(jìn)行自交,按單株收獲,T2抗性穩(wěn)定材料占19.8～32.1%,基本符合一對雜合基因分離的規(guī)律。5、田間草甘膦抗性水平和莽草酸含量結(jié)果表明,轉(zhuǎn)EPSPS-G6基因棉花種質(zhì)系可抗3倍以上的推薦濃度,其抗性水平明顯優(yōu)于美國抗草甘膦品種和本實(shí)驗(yàn)室創(chuàng)制的第一代抗草甘膦棉花種質(zhì)系,其中EPSPSG6-501種質(zhì)系抗草甘膦的能力最強(qiáng)。6、轉(zhuǎn)EPSPS-G6基因棉花種質(zhì)系籽棉產(chǎn)量、皮棉產(chǎn)量、衣分均與中棉所49近似；鈴重、籽指略高于對照；株高與對照近似；果枝數(shù)略低于對照；單株鈴數(shù)與對照一致。纖維品質(zhì)檢測結(jié)果表明,轉(zhuǎn)EPSPS-G6基因棉花種質(zhì)系的纖維長度和整齊度略優(yōu)于對照；馬克隆值與對照在同一品級且略好于對照；伸長率略低于對照；斷裂比強(qiáng)度與對照相同。說明外源基因?qū)τ谵D(zhuǎn)基因棉花種質(zhì)系的產(chǎn)量、農(nóng)藝性狀和纖維品質(zhì)無明顯影響。7、調(diào)查了金華地區(qū)的雜草發(fā)生情況與防控成本,為抗草甘膦棉花品種的應(yīng)用提供依據(jù)。結(jié)果表明,金華地區(qū)棉田雜草以牛筋草、馬齒莧和小飛蓬為主,化學(xué)除草可節(jié)省植棉除草用工開支約1000元/畝,抗草甘膦棉花品種的生產(chǎn)應(yīng)用可取得顯著的經(jīng)濟(jì)效益,具有廣闊的生產(chǎn)應(yīng)用前景。
[Abstract]:Cotton is the only crop that can produce fiber by seed. Cotton production needs more labor force. Due to the transfer of labor force, the labor price of our country is rising, and the comparative benefit and economic benefit of cotton planting are decreased. In recent years, cotton production in China has been declining year after year. The cotton production in China is still dominated by manual work, among which weeding in cotton fields is one of the main contents of cotton planting. Glyphosate of glyphosate is a typical broad-spectrum, endo-absorption-conductive herbicide. In this study, EPSPS-G6, a glyphosate resistant herbicide gene with independent intellectual property rights, was constructed into plant expression vector. Using Agrobacterium tumefaciens transformation method, the target gene was transferred to Zhongmiansuo 49, a upland cotton extension variety, to create germplasm lines with high resistance to glyphosate, and to analyze its resistance and related agronomic characters. The main results are as follows: 1, using the expression vector pCAMBIA1300-EPSPS-G6 constructed in our laboratory. The target gene EPSPS-G6 was digested and ligated with empty vector pCAMBIA2301 to construct pCAMBIA2301-EPSPS-G6. The NPII screening marker gene was removed from the original vector and the target gene was used as the screening marker. 2. The transformation method of Agrobacterium tumefaciens was optimized. The conversion solution containing 50 mg / L acetosyringone AS) could increase the positive rate to 22.18, 2.28 times as much as that of the control group (9.72%). There were significant differences in transformation efficiency among different transformation times, and the highest positive rate was obtained at 12: 00: 14: 00 at noon on the flowering day. At 13: 00 in the afternoon, 15: 00 followed by 18.860.The conversion efficiency of 8: 00 to 10: 00 in the morning was the lowest, 11.29 and 3, and the optimized transformation method of Agrobacterium tumefaciens in vivo was adopted. The EPsPS-G6 gene was introduced into the upland cotton cultivar Zhongmiansuo 49, and the transformed seeds were harvested and transformed by spraying glyphosate with 20mm glyphosate. The positive rate was 22.18%. 35 transgenic plants containing EPSPS-G6 and glyphosate resistant to glyphosate were obtained by PCR. The percentage of resistance segregation of T1 transgenic progenies ranged from 1 / 4 to 3 / 4. For T1 positive plants, 19.832. 1% of the resistant stable materials were harvested per plant. The results of glyphosate resistance level and shikimic acid content showed that transgenic cotton germplasm lines with EPSPS-G6 gene could resist more than 3 times the recommended concentration. Its resistance level was obviously superior to that of the American glyphosate resistant variety and the first generation of glyphosate resistant cotton germplasm developed in our laboratory, and the EPSPSG6-501 germplasm line had the strongest resistance to glyphosate. The yield of seed cotton, lint yield and lint percentage of cotton germplasm lines with EPSPS-G6 gene were similar to those of Zhongmiansuo 49. The boll weight and seed index were slightly higher than those of the control. Plant height was similar to that of control. The number of fruit branches was slightly lower than that of the control. The results of fiber quality test showed that the fiber length and uniformity of cotton germplasm lines with EPSPS-G6 gene were slightly better than that of the control. The micronaire value was in the same grade as the control and was slightly better than that of the control. The elongation was slightly lower than that of the control. The breaking ratio intensity was the same as that of the control. The results showed that exogenous gene had no significant effect on yield, agronomic characters and fiber quality of transgenic cotton germplasm lines. The occurrence and control cost of weeds in Jinhua area were investigated. The results showed that the main weeds in cotton field in Jinhua area were Herba canola, Portulaca oleracea and Prunus vulgaris, and chemical weeding could save about 1000 yuan per mu in the cost of weeding and weeding. The production and application of glyphosate resistant cotton varieties can obtain remarkable economic benefits and have a broad prospect of production and application.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S562

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