發(fā)布時(shí)間：2018-06-16 00:51

  本文選題：小花堿茅 + 遺傳轉(zhuǎn)化　； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：鉀是植物體內(nèi)第二大礦物營(yíng)養(yǎng)元素,它對(duì)植物生長(zhǎng)和作物生產(chǎn)至關(guān)重要。鹽堿化是影響農(nóng)業(yè)生產(chǎn)和生態(tài)環(huán)境的重要因素,土地鹽堿化已成為我國(guó)農(nóng)業(yè)生產(chǎn)發(fā)展的主要限制因素之一。由于Na+和K~+在結(jié)構(gòu)和化學(xué)性質(zhì)方面的相似性,植物體內(nèi)過(guò)多Na+的積累可干擾對(duì)K~+的吸收,抑制植物生長(zhǎng)發(fā)育甚至導(dǎo)致植物死亡。自然界中的鹽生植物在長(zhǎng)期抵御外界鹽堿脅迫的過(guò)程中形成了特殊的適應(yīng)機(jī)制,深入系統(tǒng)研究這些植物的耐鹽機(jī)理,篩選和培育適應(yīng)鹽土環(huán)境的鹽生植物,對(duì)于鹽堿土地生態(tài)系統(tǒng)的改善及土地資源的可持續(xù)發(fā)展與利用具有深遠(yuǎn)的意義。小花堿茅作為典型的拒鹽型鹽生植物,其主要耐鹽機(jī)制為限制根系單向Na+內(nèi)流,減少Na+的凈積累以維持較強(qiáng)的K~+/Na+選擇性�？梢�,小花堿茅具有極強(qiáng)的調(diào)控體內(nèi)K~+、Na+平衡的能力。但是對(duì)于小花堿茅K~+、Na+吸收與轉(zhuǎn)運(yùn)體系的系統(tǒng)性研究并不多。本研究以小花堿茅為材料,建立了小花堿茅遺傳轉(zhuǎn)化體系;采用RT-PCR等方法克隆了KT/HAK/KUP家族高親和性K~+轉(zhuǎn)運(yùn)蛋白編碼基因PtHAK5,并通過(guò)酵母異源表達(dá)試驗(yàn)驗(yàn)證了該蛋白的功能,主要取得以下結(jié)果:1.優(yōu)化了小花堿茅高頻再生體系:(1)誘導(dǎo)愈傷組織的最佳激素組合為5.0 mg·L-1 2,4-D和0.5 mg·L-1 KT,繼代過(guò)程中降低2,4-D的含量(2.5 mg·L-1)有利于提高愈傷分化率;0.5 mg·L-1 NAA、2.0 mg·L-1 6-BA和2.0 mg·L-1 KT能顯著提高幼苗的再生率,為小花堿茅愈傷分化的最佳激素組合。(2)適量有機(jī)添加物(100 mg·L-1水解酪蛋白、500 mg·L-1脯氨酸和300 mg·L-1谷氨酰胺)和0.3%的植物凝膠能有效防止愈傷玻璃化,顯著提高了胚性愈傷率。2.建立了小花堿茅高效遺傳轉(zhuǎn)化體系:(1)將繼代培養(yǎng)3-4次的愈傷組織,在添加10 g·L-1葡萄糖和200μM·L-1 AS的MS培養(yǎng)基上預(yù)培養(yǎng)4 d,其細(xì)胞結(jié)構(gòu)有利于外源DNA的整合,愈傷抵御農(nóng)桿菌侵染傷害的能力強(qiáng),侵染后轉(zhuǎn)化體的恢復(fù)能力強(qiáng)。(2)將預(yù)培養(yǎng)后的愈傷組織用OD600為0.6的GV3101農(nóng)桿菌菌液侵染15min,然后在添加200μM·L-1 AS的MS培養(yǎng)基上共培養(yǎng)2 d,為小花堿茅遺傳轉(zhuǎn)化的最優(yōu)侵染組合條件。(3)將共培養(yǎng)2 d的愈傷投入含250 mg·L-1 Cef的溶液中浸洗20 min,然后接種于添加250 mg·L-1 Cef的MS培養(yǎng)基上培養(yǎng),抑菌效果好;脫菌后,用含50mg·L-1 Kan的分化培養(yǎng)基篩選抗性愈傷。3.從小花堿茅中克隆了高親和性K~+轉(zhuǎn)運(yùn)蛋白編碼基因PtHAK5,ORF框長(zhǎng)2355 bp,推測(cè)編碼784個(gè)氨基酸,具有11個(gè)跨膜區(qū),與其他高親和性K~+轉(zhuǎn)運(yùn)蛋白具有較高的同源性,為KT/HAK/KUP家族第Ⅰ亞族成員。4.PtHAK5的超表達(dá)能夠恢復(fù)K~+吸收功能缺失的酵母突變菌株CY162在低K~+條件下的生長(zhǎng),而不會(huì)增加Na+外排功能缺失的突變菌株G19的鹽敏感性,表明PtHAK5只介導(dǎo)K~+吸收,不參與Na+轉(zhuǎn)運(yùn)。
[Abstract]:Potassium is the second most important mineral nutrient in plants, which is very important for plant growth and crop production. Salinization is an important factor affecting agricultural production and ecological environment. Land salinization has become one of the main limiting factors for the development of agricultural production in China. Because of the similarity between Na and K ~ in structure and chemical properties, excessive Na accumulation in plants can interfere with the absorption of K ~, inhibit the growth and development of plants and even lead to plant death. Halophytes in nature have formed special adaptation mechanism in the process of resisting external saline-alkali stress for a long time. The mechanism of salt tolerance of these plants is studied systematically and the halophytes adapted to salt soil environment are screened and cultivated. It is of great significance for the improvement of saline-alkali land ecosystem and the sustainable development and utilization of land resources. As a typical salt-repellent halophyte, the main salt tolerance mechanism of Festuca florescens is to limit the one-way Na influx of roots and reduce the net accumulation of Na to maintain strong K- / Na selectivity. It can be seen that Flos lanceolata has a strong ability to regulate K ~ + Na balance in vivo. But there are few systematic studies on K ~ + Na absorption and transport system. In this study, the genetic transformation system of Festuca flosiflora was established, and the gene PtHAK5 of KT / HAKR / KUP family was cloned by RT-PCR, and the function of PtHAK5 was verified by yeast heterologous expression test. The main results are as follows: 1. The optimal hormone combinations for callus induction were 5.0 mg L ~ (-1) 2H ~ (4-D) and 0.5 mg ~ (-1) KT, and the decrease of 2H _ 4-D content was 2.5 mg / L ~ (-1) during subculture.) the callus differentiation rate was increased by 0.5 mg 路L ~ (-1) NAA ~ (2 mg 路L ~ (-1) 6-BA and 2.0 mg ~ (-1) KT. Increase the regeneration rate of seedlings, It is the best hormone combination for callus differentiation of Flos alkaloids. 2) 100 mg L-1 hydrolysate of casein 100 mg L-1 proline and 300 mg L-1 glutamine) and 0.3% plant gel can effectively prevent vitrification of callus and significantly increase the rate of embryogenic callus. The callus was subcultured for 3-4 times and precultured on MS medium supplemented with 10 g L-1 glucose and 200 渭 M L-1 as for 4 days. The cell structure was beneficial to the integration of exogenous DNA. The ability of callus to resist the infection injury of Agrobacterium tumefaciens is strong. The precultured callus was infected with GV3101 Agrobacterium tumefaciens solution with OD600 0.6 for 15 min, and then co-cultured on MS medium supplemented with 200 渭 M L-1 as for 2 days. The callus co-cultured for 2 days was soaked in 250 mg L-1 Cef solution for 20 min and then cultured on MS medium supplemented with 250 mg L-1 Cef. The antimicrobial effect was good, and the resistant callus was screened by the differentiation medium containing 50mg L-1 Kan. The high affinity K ~ transporter encoding gene PtHAK5 ORF was cloned from Festuca chinensis with a length of 2355 BP, presumably encoding 784 amino acids, with 11 transmembrane regions, with high homology with other highly compatible K ~ transporters. The overexpression of KT / Hak / KUP family member. 4. PtHAK5 overexpression could restore the growth of yeast mutant CY162 without increasing the salt sensitivity of the mutant strain G19, which was deficient in K ~ absorption function, but could not increase the salt sensitivity of mutant strain G19, which was deficient in Na efflux function. PtHAK5 only mediates K ~ absorption and does not participate in Na transport.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S543.9

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