【作者】 范明霞；

【导师】 罗利军； 刘灶长；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 木薯作为食品以及饲料以及加工业的原料,在市场上占有的份额已经越来越大。但是由于木薯自身性状的原因制约了传统常规育种的发展。而木薯易发生采后生理性变质,易受病毒病危害,不耐寒,叶片早衰、植株含有毒性氰化物等性状很难通过传统育种的方法获得改良,因此诱变育种、基因工程育种、分子标记辅助选择育种渐渐进入了科学家的视野。本文建立了一套高效的植株再生和遗传转化体系,以便于开展木薯基因工程育种。本研究通过这个体系将克隆的生氰糖苷裂解酶HNL24b基因转化木薯,以减少植株中的氰化物含量,并期望通过块根特异性启动子patatin启动该基因的表达,从而达到使得木薯根叶中的氰化物含量有所不同的目的。同时也对木薯辐照诱变育种进行研究,并从中筛选到品质性状优良的突变株系。主要研究结果如下：1.建立微型薯再生体系。研究发现培养基中添有0.5 mg/L的NAA最有利于木薯茎的生长和须根的形成,添加0.5 mg/L BA和0.5 mg/L NAA有利于微型薯的形成。在初生培养阶段生长出须根最多的植株经第二阶段培养后结薯更容易。在添加0.5mg/LBA和0.5 mg/L NAA的条件下,6%(M／V)蔗糖对植株生长最有利,并在得芽率(62.34%)、株高(5.6044 cm)、微型薯得率(71.42%)、平均薯鲜重(0.6129g)上优于其它处理。2.研究不同激素以及光照条件对愈伤形成的影响。研究发现添加1mg/L KT和5 mg/L NAA有利于木薯幼嫩叶片形成愈伤；前期经8 h/d光照有利于愈伤的形成；前期经0 h/d光照有利于愈伤的绿化；前期培养,在0 h/d光照条件下,并在培养基中加有1 mg/L KT和2 mg/L NAA时生根率最高,可以达到97.51%。3.通过胚状体形成途径研究了木薯的再生体系,结果表明添加12 mg/L的picloram对于木薯胚状体的形成、胚性子叶再生以及植株的再生最有利,其再生频率分别为97.53%、98.72%、44.16%。Picloram比2,4-D和NAA更适于诱导木薯胚状体。10 mg/L的picloram更加适用于诱导次生胚状体,其诱导频率高达95.76%。低浓度的BA有利于胚性子叶的再生,高浓度的BA将促使非胚性子叶的形成。当BA浓度为0.1 mg/L时,胚性子叶的再生频率最高,可达95%。培养基添加1 mg/L的BA与1 mg/L的IBA有利于植株的再生,其频率高达47.60%,优于BA与IAA和NAA的其它配比。1 mg/L的BA与1 mg/L的NAA有利于子叶胚生根的。4.探讨了用不同的方法提取木薯的RNA,研究发现用改良CTAB与LiCl相结合的方法,可以提取到质量较好的RNA。5.本研究成功克隆到HNL24b基因,本研究克隆的MeHNL24b序列与MeHNL4的氨基酸同源性达87.72%,与HbHNL的氨基酸同源性达86.32%,与HNL24的氨基酸同源性达42.81%。预测该基因编码的蛋白中1-15位氨基酸区域属于信号肽区域,31-252位氨基酸区域属于水解酶特有的α/β折叠结构域。该蛋白具有水解酶的活化中心位点：丝氨酸,天门冬氨酸和谷氨酸(ser/asp/glu)。预测此编码蛋白域的组成元素有：1344个C、2091个H、343个N、379个O、7个S。6.成功从马铃薯中克隆到块根特异表达启动子Patatinb,它与已报道的patatin启动子序列有95%的相似性。7.成功构建原核表达载体,并获得有功能的表达蛋白,经酶活测定HNL24b的最高酶活力为2978 U/L。8.成功构建HNL24b基因的超表达载体、反义表达载体以及GUS表达载体,并获得转化子；成功构建了块根特异表达载体。HNL24b基因在转基因植株中的表达,能有效的减少植株中氰化物的含量。9.通过辐照诱变育种,获得了具有一个或多个优良性状的木薯突变株系。调查的性状包括产量、株高、赖氨酸含量、HNL表达量、采后生理变质程度。更多还原

【Abstract】 Cassava as food, feed and raw materials of processing industry, its share of the market has been growing. However, due to its characteristics, the development of conventional breeding is limited. Cassava have some important traits, such as physiological post harvest deterioration, Vulnerable to virus damage, prematurely leaves senile and plants containing cyanide and so on. It is hard to modify these traits through traditional breeding, so scientists are paying attention to mutation breeding, genetic engineering, molecular markers assisted selection. In this paper, an efficient plant regeneration and transformation system had been established. In order to more efficiently express HNL and reduce the cyanide content in the plants, the full length DNA of HNL24b was cloned and over expressed in cassava. In order to make the roots and leaves have different cyanide content, a root specific promoter patatin had been cloned from potato and used for activating HNL24b gene expression specifically in roots. Also, the irradiation technique for inducing mutants with fine quality traits of cassava was investigated. The main results briefly were as follows:1. Adding 0.5 mg/L of alpha-naphthalene acetic acid (NAA) to the MS based medium was most favorable to the in vitro growth of cassava stem and fibrous roots. Combination of 0.5 mg/L BA and 0.5mg/L NAA was most favorable to the growth of micro-tubers. BA (0 mg/L-2.0 mg/L) showed effective on shoot regeneration. NAA (0 mg/L-2.0 mg/L) proved to be effective on root development. Plantlets with fibrous roots turned out to be easy to generate microtubers in vitro. The microtubers were inducted only when both BA and NAA were used in combination. MS medium supplemented with sucrose at 6%(w/v) resulted in the highest frequencies of shoot induction (62.34%), average shoots height (5.6044 cm), microtuber induction (71.42%) and average fresh weight of microtubers (0.6129 g), all superiors to other treatments, when BA (0.5 mg/ L) and NAA (0.5 mg/L) were also added to the MS medium.2. Addition of 1 mg/L KT and 5 mg/L NAA to MS medium showed effective on formation of cassava leaf callus. Early culture with 8 h/d light was beneficial to the growth of callus. Early culture with 0 h/d light was beneficial for the callus to grow into green. Early culture with Oh/d light and 1 mg/L KT and 2 mg/L NAA was beneficial to rooting, with the rooting rate being 97.51%.3. Addition of 12 mg/L of picloram resulted in the highest rate of somatic embryogenesis (97.53%), cotyledon regeneration (98.72%) and plant regeneration (44.16%). It indicated that picloram was more suitable than 2,4-D and NAA for cassava somatic embryogenesis.10 mg/L of picloram is more suitable for induction of secondary embryoids (95.76%). Low concentration of BA was most favorable to the regeneration of embryogenic cotyledons, while high concentration of BA will lead to the growth of non-embryonic cotyledon. As addition of BA at 0.1 mg/L, frequency of embryogenic cotyledon regeneration reached the highest at 95%.Addition of 1 mg/L of BA and 1 mg/L of IBA resulted in the highest frequencies of plant regeneration (47.6%). 1mg/L of BA and 1 mg/L of NAA showed effective on rooting of embryogenic cotyledons.4. The CTAB method combined application of LiCl turned out to be a good protocol for cassava RNA extraction with good quality.5. The full length cDNA of HNL24b gene was obtained by RT-PCR cloning. The sequencing result for the DNA showed that the sequence encoding for the HNL was not fully consistent with those already published. The full sequences analysis demonstrated that the highest homology of amino acid sequence about 87.72% to MeHNL4 genes, about 86.32% to HbHNL and about 42.81% to MeHNL24. The cDNA was cloned into expression vectors pBI121, pCAMBIA 1323, PET32a(+) by PCR and recombinant DNA technique. Prediction of the encoding protein indicated that the 1-15 amino acids were signal peptide region,31-252 amino acids wereα/βfold domain region. Predicted elements composition of this protein is as follow:C (1344), H (2091), N (343), O (379), S(7).6. The Solanum tuberosum tuber specific promoter Patatinb was cloned. The full sequences analysis demonstrated that the highest homology of sequence was about 95% to Patatin genes from Solanum tuberosum.7. After the transformation of PET32a(+), screening for high copy transformants, induction with SDS-PAGE analysis, enzymatic activityassay, the HNL24b efficiently expressed in BL21 and the enzyme activity reached over 2978 units/L of culture product.8. HNL24b gene was inserted into expression vector, antisense expression vector, GUS expression vector and transformants of those vectors were obtained. A root-specific expression vector was constructed. Expression of HNL24b gene in transgenic plants can effectively reduce the cyanide content in plants.9. By Gama ray irradiation, cassava mutants with one or more good traits, such as high yield, high content of lysine, high HNL expression, lower post-harvest physiological deterioration were obtained.更多还原