PEPc基因RNAi的擬南芥轉(zhuǎn)化及性狀分析

生物工程論文編號:SW067  論文字?jǐn)?shù):11820,頁數(shù):27

目　錄

中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
1. 緒 論 1
 1.1  油料作物基因工程研究進(jìn)展 1
 1.2  磷酸烯醇式丙酮酸羧化酶（PEPc）基因 2
 1.3  RNAi研究現(xiàn)狀 2
 1.4  模式植物擬南芥中的PEPc基因 3
 1.5  本課題的研究目標(biāo)及意義 4
2. 實(shí)驗(yàn)部分 5
 2.1  農(nóng)桿菌介導(dǎo)的擬南芥轉(zhuǎn)化 5
 2.2  轉(zhuǎn)基因擬南芥的篩選與鑒定 7
 2.3  轉(zhuǎn)基因擬南芥植株的性狀分析 9
3. 結(jié)果與討論 11
 3.1  農(nóng)桿菌介導(dǎo)的擬南芥轉(zhuǎn)化 11
 3.2  實(shí)驗(yàn)結(jié)果分析 11
 3.2  PEPi轉(zhuǎn)基因擬南芥的篩選與鑒定 12
 3.3  轉(zhuǎn)基因擬南芥植株的性狀分析 14
4. 總結(jié)與展望 19
致謝 20
參考文獻(xiàn) 21

摘 要：利用基因工程技術(shù)進(jìn)行植物高油育種，是當(dāng)前國際上植物基因工程研究的熱點(diǎn)之一。找到控制作物種子含油量的相關(guān)基因，通過運(yùn)用生物技術(shù)，抑制或超表達(dá)目的基因，探討該基因在植物中的調(diào)控機(jī)理，已成為此類研究的主要方法。作物種子中的貯藏物質(zhì)主要由蛋白質(zhì)、油脂及糖類組成，在油料作物中蛋白質(zhì)及油脂構(gòu)成其干物質(zhì)的70％以上。研究表明作物種子中磷酸烯醇式丙酮酸羧化酶（phosphoenopyruvate carboxylase,簡稱PEPc）活性與其蛋白質(zhì)含量正相關(guān)，而與油脂含量呈負(fù)相關(guān)。為了探討PEPc基因調(diào)控油分/蛋白的分子機(jī)理，本實(shí)驗(yàn)通過構(gòu)建好的PEPc基因RNAi的載體（命名為PEPi），通過花粉管浸泡法將PEPi載體導(dǎo)入擬南芥，對轉(zhuǎn)化后的擬南芥進(jìn)行除草劑抗性篩選、PCR鑒定得到陽性轉(zhuǎn)基因植株，并利用除草劑篩選統(tǒng)計(jì)了轉(zhuǎn)基因擬南芥T2代的遺傳分離比，大部分符合3:1的經(jīng)典遺傳分離比。利用氣相色譜分析測定T3代轉(zhuǎn)基因植株種子含油量，表明轉(zhuǎn)基因可以提高種子含油量，在鹽處理的初步實(shí)驗(yàn)中還觀察到轉(zhuǎn)基因種子的抗鹽性有明顯降低。
關(guān)鍵詞： PEPc基因；擬南芥(Arabidopsis thaliana)；RNAi；含油量

Abstract: The use of genetic engineering technology for high oil plant breeding was the one of the hot international plant genetic engineering at current. Through the use of biotechnology to inhibit gene expression or enhance gene expression in the plant, the relevant gene of the controlling crop seed oil was found. Crop seeds store material mainly from protein, fat and carbohydrate, protein and fat constitute over 70% of the dry matter in Oil-seed composition. Research shows that phosphoenopyruvate carboxylase (PEPc) which activity was relevant with protein content and negatively correlated with fat content in crop seeds. To explore PEPc gene how to regulate oil / protein in molecular mechanism, this experiment has set the PEPc gene of RNAi vector up and carried it into Arabidopsis through tube with the method of tube immersion. Then the herbicide resistance of the transformed Arabidopsis screening was measured. In result, positive strains of transgenic plants were obtained through PCR identification. Statistic from transgenic Arabidopsis T2 generation which screened by herbicide resistance showed that it was segregation ratio most with a 3:1 classic genetic ratio. Using gas chromatographic analysis for T3 transgenic plant seed oil content, it was showed that the transgenic seeds can increase oil content.  In the salt preliminary experiment which also observed that the transgenic seeds were significantly reduced in resistance salt.

Keywords : PEPc gene；Arabidopsis thaliana； RNAi；Oil content