摘要
胡枝子(Lespedeza Michx.)是优良的水土保持和饲料型灌木,提高其抗逆性和饲用品质存农业、林业生产上具有重要意义。
许多转基因成果表明外源甜菜碱醛脱氢酶(BADH)基因的导入可提高植物的抗旱、耐盐性。4-香豆酸-辅酶A-连接酶(4CL)是木质素合成过程中的关键酶,反义转入4CL基因可降低一些植物的木质素。木文选用BADH基因、反义4CL基因为目的基因,构建了以木糖异构酶(xylA)基因为标记基因的植物表达载体pBI121-xylA-BADH和无选择标记基因的双元植物表达载体反义4CL-BADH,期望通过转基因技术实现胡枝子木质素和抗逆性的遗传改良。试验材料选用生物量大,来自美国的二色胡枝子(Lespedeza bicolor Turcz.)为研究对象,优化了二色胡枝子的再生体系,建立了二色胡枝子遗传转化中的木糖选择系统和无标记基因选择系统,并将xylA-BADH基因、反义4CL-BADH基因转入到二色胡枝子之中,获得了转基因植株,并检测了转基因植株的部分功能。该项研究对促进转基因胡枝子的生产应用和胡枝子饲料产业的发展具有理论和现实意义。
主要研究内容和结果如下:
(1)从大肠杆菌基因组中克隆出了木糖异构酶基因xylA,构建了以木糖为选择剂的植物表达载体pBI121-xylA-BADH和无选择标记基因的双元植物表达载体反义4CL-BADH,并将这两个表达载体转化到根癌农杆菌中。
(2)在二色胡枝子再生体系建立和优化过程中,确定了二色胡枝子培养过程中的基本培养基为大量元素浓度减半的MS培养基,命名为MS*。确定了二色胡枝子子叶节分化最适配方为:MS*+6-BA 1-0 mg.L~(-1)+NAA 0.2 mg.L~(-1)+蔗糖3%+琼脂6 g.L~(-1),茎段增殖适合配方为:MS*+6-BA 1.0 mg.L~(-1)+NAA 0.1 mg.L~(-1)+IBA 0.2 mg.L~(-1)+蔗糖3%+琼脂6 g.L~(-1);生根最适配方为:MS*+NAA 0.1 mg.L~(-1)+IBA 0.2 mg.L~(-1)+蔗糖3%+琼脂6 g.L~(-1)。
(3)确定了二色胡枝子木糖选择系统和无标记基因选择系统的建立中头孢霉素的适用浓度,即:子叶节分化过程中为200～300 mg.L~(-1),茎段增殖和生根过程中为100 mg.L~(-1)。
当用木糖为选择剂进行pBI121-xylA-BADH遗传转化时,以3%的木糖取代蔗糖为子叶节不定芽分化过程中的选择剂,以0.5%的蔗糖和2.5%的木糖混合碳源作为抗性芽增殖和生根过程的选择剂。
在反义4CL-BADH对二色胡枝子进行遗传转化研究中,确定了在培养基中添加1g.L~(-1)的甜菜碱醛作为子叶节分化中的选择剂,培养基中添加0.3g.L~(-1)的甜菜碱醛和0.7g.L~(-1)的NaCl作为抗性芽增殖和生根过程的选择剂。
(4)经过PCR、PCR-Southern和Southern检测,获得了转入xylA-BADH基因的二色胡枝子和转入反义4CL-BADH基因的二色胡枝子。
耐盐性试验表明转入反义4CL单基因的二色胡枝子耐盐性最差,而转入xylA-BADH基因的二色胡枝子耐盐性最强,转入反义4CL-BADH双价基因的二色胡枝子耐盐性略次于非转基因材料。转入反义4CL-BADH基因的二色胡枝子组培苗经过1.0g.L~(-1)NaCl胁迫培养,其甜菜碱醛脱氢酶活性可达对照材料的2.5倍。
通过M(a｜¨)ule反应、Weisner反应、卢索夫法组织化学染色和木质素荧光观察,初步表明反义4CL-BADH基因的导入有可能降低二色胡枝子的木质素含量,并对检测方法的可靠性进行了讨论。
此外,转入反义4CL-BADH的二色胡枝子茎段皮层组织中含有的异形细胞数量明显比对照多。通过对异形细胞、植物抗逆性、甜菜碱三者之间的关系讨论,认为转基因植株异形细胞数量多是由于BADH基因的导入可能合成了较多甜菜碱。
Lespedeza Michx.plants are excellent fodder shrubs and also trees which can be used to conserve soil and water.It's important on improving their tolerance to abiotic stress in agricultural and forestry production.
Many transgenic results showed that the transgenic plants carring betaine aldhyde dehydrogenase(BADH) gene could improve their tolerance to drought or salt stress. 4-coumarate:CoA ligase(4CL) is the key enzyme in the process of lignin synthesis. Transgenic plants with antisense 4CL gene reduced the lignin content.In this paper,the BADH & antisense 4CL genes were selected as objective genes.The pBI121-xylA-BADH plant expression vector with xylose being the selective agent was built.Another plant expression vector named antisense 4CL-BADH was built too.We expect to use gene engineering to improve the characters of Lespedeza in the aspect of the lignin content and abiotic resistance.Lespedeza bicolor Turcz.from America which has abundant harvest was selected as the research object.The high frequency regeneration system of L.bicolor was optimized.L.bicolor genetic transformation system based on xylose as the selective agent was built,and the marker free transgenic system was built too.The xylA-BADH genes and antisense 4CL-BADH genes were introduced into L.bicolor.Some functions of transgenic plants were assayed.The research findings will have theoretic & real significance on promoting the development of fodder industry which Lespedeza Michx.plants are used as fodder.
The main research work and results are as follows:
(1) The xylose isomerase(xylA) gene was cloned from E.coli.The pBI121-xylA-BADH plant expression vector with xylose being the selective agent was built. Another new plant expression vector without marker gene named antisense 4CL-BADH was built too.The plasmid DNA of pBI121-xylA-BADH and antisense 4CL-BADH were transformed into LBA4404,EHA105 individually.
(2) During the building and optimization of regeneration system of L.bicolor,the main medium named MS~* with having a half of the macronutrients in MS medium was used.The optimal medium for adventitious buds regeneration of L.bicolor cotyledonary node was MS~* + 6-BA 1.0 mg.L~(-1) + NAA 0.2 mg.L~(-1) + sucrose 3%+ agar 6 g.L~(-1).The optimal medium for segment proliferation was MS~* + 6-BA 1.0 mg.L~(-1) + NAA 0.1 mg.L~(-1) + IBA 0.2 mg.L~(-1) + sucrose 3%+ agar 6 g.L~(-1).The optimal medium for rooting of L. bicolor plants in vitro was MS~* + NAA 0.1 mg.L"l + IBA 0.2 mg.Lt + sucrose 3%+ agar 6 g.L~(-1).
(3) The agrobaeterium-mediated xylose selective system and marker free selective system of L.bicolor were studied.200～300 mg.L~(-1) Cefotaxine(Cef) could restrain the growing of agrobacterium during the adventitious buds regeneration of L.bicolor cotyledonary node.100 mg.L~(-1) Cef was enough to restrain agrobacterium during proliferation or rooting.
When pBI121-xylA-BADH plasmid DNA was introduced into L.bicolor,3%xylose could be the selective agent at adventitious buds regeneration of cotyledonary node and 0.5%sucrose & 2.5%xyiose could be the selective agent at proliferation or rooting.
The methods of introducing antisense 4CL-BADH into L.bicolor were studied,1g.L~(-1) betaine aldehyde chloride was the selective agent for adventitious buds regeneration of cotyledonary node.The mixture of 0.3 g.L~(-1) betaine aldehyde chloride & 0.7 g.L~(-1) NaCl was the selective agent for proliferation or rooting.
(4) After PCR,PCR-Southern and Southern assays had been done,xylA-BADH and antisense 4CL-BADH were sure to be introduced into L.bicolor.
The study on tolerance to salt stress showed that the xylA-BADH transgenic plants had the best tolerance and the antisense 4CL transgenic plants were the worst.The tolerance of antisense 4CL-BADH transgenic plants to salt stress was slight lower than wild-type plants. After being cultivated for some days at 1.0g.L~(-1) NaCl stress,the activity of betaine aldehyde dehydrigenase in antisense 4CL-BADH transgeinic plants was 2.5 times than that of in the wild-type plants.
The stem slices of L.bicolor were observed by the histochemical staining of M(a|¨)ule responses,Weisner responses,Rosoff responses and fluorescence of lignin responses.The preliminary result showed that the transgeinic L.bicolor with antisense 4CL-BADH maybe could reduce the lignin content.The reliability of the assay methods about the antisense 4CL transgenic plants was discussed.
In addition,the number of idioblastic cells in the stem slices of transgeinic L.bicolor with antisense 4CL-BADH was more than those of wild-type L.bicolor.After discussion of the relationship among idioblastic cells,tolerance to abiotic stress and betaine,the author thought the reason of transgenic plants having an increased number of idioblastic cells was that BADH was introduced and much betaine might be synthesized.
引文
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