摘要
花生(Arachis hypogaea L.)起源于南美洲,是世界第四大含油种子作物。花生油主要由棕榈酸,油酸和亚油酸组成。油类中多不饱和脂肪酸的含量、组成和生理状态是油类品质最重要的决定因素。植物油富含油酸,是比富含多不饱和脂肪酸的油类更优质的产品。高油酸、低多不饱和脂肪酸的油类便于贮藏,对人体健康大有益处,是商业和营养上所需要的。Δ12-油酸脱氢酶(Δ12-oleic acid desaturase,FAD2)向脂肪酸碳链上引入第二个双键,是促进油酸转化为亚油酸的关键酶。FAD2基因开放读码框(ORF)为1140bp,编码379个氨基酸,它包含一个内含子,位于编码区3’末端。花生中存在两个FAD2同源基因(AhFAD2A, AhFAD2B),它们可能是来自原始二倍体起源种的同源基因。ahFAD2A的突变和ahFAD2B转录物水平的大量降低共同造成了花生品种中的高油酸表型,并且一个编码功能性酶的表达基因似乎对正常油酸表型是足够的。FAD2基因的表达水平还受温度影响。总之,FAD2A、FAD2B这两个同功酶基因是控制油酸含量的关键酶基因,但可能其他酶基因或多种调控机制也起着重要作用。FAD2在微生物工程、油料作物基因工程和植物油质改造等方面成为科学研究的热点之一。
本试验将倒位重复的Δ12脂肪酸脱氢酶基因AhFAD2片段与经改造的含CaMV35S启动子的表达载体pCAMBIA1302相连,构建了载体pCSF;将倒位重复的AhFAD2基因与种子特异性表达的大豆凝集素启动子相连连接到表达载体pCAMBIA1300中,构建了载体pCLF,将两个载体导入农杆菌LBA4404和EHA105,以山东主产大花生鲁花14号和丰花1号成熟种子的上胚轴和子叶为受体材料进行遗传转化,实现该倒位重复基因片段对花生中AhFAD2基因表达的抑制。由于在双子叶植物,特别是花生中,使用潮霉素做筛选剂的少有报道,且不同品种花生、花生不同部位对潮霉素浓度的敏感度差异较大,因此在进行花生的遗传转化之前,我们设计了一组梯度筛选试验。最终确定花生胚轴转化体系的筛选浓度为潮霉素25mg/L,子叶转化体系的筛选浓度为潮霉素10mg/L,即为筛选剂的基础压力。此外,不同农杆菌菌株对不同植物的转化效率差异很大。曾有报道指出农杆菌LBA4404和EHA105对花生具有较高的转化效率,为确定哪种对花生转化效率更高,我们选取了这两种菌株进行对照实验。结果显示与LBA4404 42.01%的转化率相比,EHA105对花生具有更高的转化率,可达48.74%。经潮霉素选择培养基筛选培养,获得了转化植株400余株,其中CaMV35S启动子启动的180余株,种子特异性表达的大豆凝集素启动子启动的230余株。我们对转化苗进行了分子生物学的验证,PCR结果显示,CaMV35S启动子调控表达的植株和种子特异性表达的大豆凝集素启动子调控表达的植株均有大部分扩增出372bp的特异性目的条带,表明Δ12脂肪酸脱氢酶基因AhFAD2的RNAi抑制表达框架已整合进花生中。PCR Southern结果表明,野生型植株(对照)没有明显的杂交信号,转基因株系有强阳性信号。
作为对照,我们同时培养了未经转化的再生苗。与再生植株相比经筛选获得的转化苗部分植株表现畸形,出现叶片皱缩浓绿,基部膨大,果枝间距缩短,不能正常生根等现象,其余虽形态正常但大都生长缓慢,茎秆弱小,尝试了多种方法进行改进,但效果不太理想。脂肪酸代谢是生物体基本生命活动之一,脂肪酸是膜脂结构的重要组成部分,这些现象也许是由于抑制了Δ12脂肪酸脱氢酶基因所引起的,但是也有报道指出潮霉素对组培植株的生长发育具有很多不利影响,并且花生切口深入培养基中,造成了丛生芽形成和生长过程中呼吸不畅,还有报道指出高浓度的BA抑制了原先芽点的生长及随后新芽的继续分化生长。因此,对于造成上述现象的深层原因还有待进一步研究。
Peanut (Arachis hypogaea L.) is the fourth largest oilseed crop in the world, which originated from South America. Peanut oilseeds consists mainly of palmitic, oleic and linoleic acids. The content, composition and condition of the polyunsaturated fatty acids in oilseeds are the most important determinant factors of oil quality. Plant oils rich in oleate are considered superior products compared to oils rich in polyunsaturated fatty acids. Oils with higher oleic acid and lower polyunsaturated fatty acids are good for health , and satisfy desirability commercially and nutritionally.Δ~(12)- oleic acid desaturase which places the second double bond in the fatty acid chain, is the key enzyme which desaturates oleic acid to linoleic acid. The ?12- oleic acid desaturase gene contains a putative intron, the coding region at the 3′end, and an open reading frame (ORF) of 1140 bp encoding 379 amino acids. There exist two homeologous genes(AhFAD2A, AhFAD2B) forΔ~(12)-oleic acid desaturase in peanut, which derived from the original diploid progenitor species. A mutation in ahFAD2A and significant reduction in levels of the ahFAD2B transcript together cause the high oleate phenotype in peanut varieties, and that one of them expressed a functional enzyme appears to be sufficient for the normal oleate phenotype. The expression of ?12- oleic acid desaturase gene also are affected by temperature. All in all, the two homeologous genes, FAD2A and FAD2B, are the key genes which detemine the concentration of oleic acid in peanut, although other genes or control mechanisms may also play an important role.Δ~(12)- oleic acid desaturase has become one of the major researching enzyme in the area of microbial engineering , oilseeds crop gene engineering and plant lipid modification.
We linked the inverted repeat sequence of partialΔ~(12) fatty acid desaturase gene AhFAD2 with CaMV35S promoter and inserted them into binary plant vector pCAMBIA1300, and linked the inverted repeat sequence of partial AhFAD2 gene with strong seed specific soybean Lectin promoter and inserted them into binary plant vector pCAMBIA1302. The resulting plasmid, named pCSF and pCLF, respectively, were mobilized to Agrobacterium tumefaciens strain LBA4404 and EHA105, used for plant transformation. We used hypocotyls and cotyledons of matured peanut seeds of Luhua No.14 and Fenghua No.1 as recipients, the inverted repeat sequence of partialΔ~(12) fatty acid desaturase gene AhFAD2 was introduced into Arachis hypogaea L via Agrobacterium tumefaciens infection. Because there are only a little reportes about the screening concentration of hygromicin B in dicotyledonous plants, especially in Arachis hypogaea L, furthermore, there are big sensitivity differences between different species and different part of Arachis hypogaea L, we designed a gradient screening tests before transformation. Finally, we determined the screening concentration of hygromicin B for hypocotyls is 25mg/L and for cotyledons is 10mg/L. In addition, to differnent species of plants, there are big transformation efficiency differences between different strains of Agrobacterium tumefaciens. It was reported that Agrobacterium tumefaciens strain LBA4404 and EHA105 both had high transformation efficiency to Arachis hypogaea L. In order to determine which strain had higher transformation efficiency to Arachis hypogaea L, we made a contrast experiment between the two strains. The result showed that compared with LBA4404, EHA105 had higher transformation efficiency to Arachis hypogaea L. Transformants were selected for their ability to grow on medium containing hygromicin B, more than 400 lines that were all tolerant to hygromicin B were selected, 180 of them were driven by CaMV35S promoter, and 230 of them were driven by strong seed specific soybean Lectin promoter. The transformants used for further molecular determination. PCR showed that most of the transformants had strong positive signals, and no signal was shown in wild type plants. This indicated that the inverted repeat structure had introduced into Arachis hypogaea L successfully. PCR Southern blotting analysis also indicated that the structure had been integrated into all of the transgenic plants’genomes.
As control, we cultured the regeneration plants at the same time which haven’t been transformated. Compared with regeneration plants, some of the transformants showed abnormal form, such as leaves dark green and wrinkle, the base of plant swollen, the space of branch shortened, root abnormal, and so on. Although the form of others were normal, most of them showed slow-growing and the branches of them were weak. Tried many methods to improve, but the effcets were not noticeable. Fatty acids matebolism is one of the basic vital processes, and fatty acids are imprtant component part of membranous lipids, so the phenomenons above may result from the supressed expression ofΔ12 fatty acid desaturase gene AhFAD2. And also there are reportes showed that hygromicin B has many disadvantages for plant develepoment. Thus, the reasons which result in the phenomenons above, still need further research.
引文
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