敖汉苜蓿小花与种子响应硼胁迫的蛋白质组学与代谢组学分析

发布时间：2018-06-07 01:38

本文选题：敖汉苜蓿 + 小花　；参考：《中国农业大学》2017年博士论文

【摘要】：硼是紫花苜蓿必需的微量元素之一,它能促进紫花苜蓿生殖器官的正常发育,有利于开花结实,对提高苜蓿种子产量和质量的作用不可估量。苜蓿对硼的需求在适量和过量之间的界限很小,施用不当会使苜蓿中毒从而降低种子产量。因此开展紫花苜蓿硼营养机理以及紫花苜蓿生殖器官响应硼胁迫的机制研究,有助于实际生产过程中合理使用硼肥,有效提高苜蓿种子产量。本论文在实际生产实验的基础上,以敖汉苜蓿小花和种子为实验材料,应用蛋白质组学和代谢组学技术,研究硼胁迫下蛋白质及小分子代谢产物的变化及代谢途径,探索紫花苜蓿生殖器官响应硼胁迫的应答机制。主要研究内容如下:1.选用0、0.04、0.08、0.12和0.16%浓度的硼,对生长第二年的敖汉苜蓿进行施肥实验。结果表明,施硼肥有效提高紫花苜蓿小花花粉数量、花粉活力、种子产量、产量构成因子及种子质量。总体上,0.08%浓度处理组的效果较好,增产效果显著。而0.12%处理组存在落花现象,从而降低种子产量。当喷施浓度达0.16%时,已产生毒害作用。因此选择0.00%浓度作为缺硼处理,0.08%浓度作为最适施肥量,0.16%浓度作为高硼处理进行下一步研究。2.测定了 0%(缺硼)、0.08%(对照)和0.16%(高硼)硼处理的敖汉苜蓿小花与种子的抗氧化酶活性、MDA、脯氨酸和可溶性糖含量。测定结果表明,在缺硼胁迫下敖汉苜蓿小花与种子的抗氧化酶活性、MDA和脯氨酸含量均有不同程度的下降。在高硼处理下敖汉苜蓿种子抗氧化酶活性和MDA含量增加,而脯氨酸和可溶性糖含量下降。高硼胁迫对敖汉苜蓿小花的影响不大,但对种子造成一定的影响。3.运用2-DE技术对0%、0.08%和0.16%硼处理的敖汉苜蓿小花与种子蛋白质进行分离,并用PDQuest图像软件、LC-MS/MS质谱技术、Mascot软件和Uniprot数据库进行分析鉴定。在缺硼胁迫下从敖汉苜蓿小花中成功鉴定出12个差异蛋白,主要参与碳水化合物代谢和脂质代谢。从种子中成功鉴定出11个差异蛋白,主要参与碳水化合物代谢和氨基酸代谢。在高硼胁迫下从敖汉苜蓿小花中共筛选出14个差异蛋白,主要参与碳水化合物代谢、氨基酸代谢、脂质代谢和次生代谢。从种子中共筛选出11个差异蛋白,主要参与了氨基酸代谢、碳水化合物代谢和脂质代谢。敖汉苜蓿小花酰基蛋白硫酯酶在缺硼和高硼胁迫下均下调表达,敖汉苜蓿种子NAD依赖的醛脱氢酶家族蛋白在缺硼和高硼胁迫下均上调表达。NAD(P)结合罗斯曼折叠蛋白在高硼处理的紫花苜蓿小花与种子中均表达,是紫花苜蓿响应硼毒害的关键调控蛋白。4.利用GC-MS技术,对缺硼和高硼胁迫下敖汉苜蓿小花与种子进行代谢物谱分析。采用OPLS-DA模型第一主成分的VIP值(阈值1)结合单维统计(t检验)的p值(阈值p0.05)寻找差异性表达代谢物。在缺硼胁迫处理组中共筛选到50个差异物质,主要涉及碳水化合物代谢和氨基酸代谢;从种子中共筛选到13个差异物质,主要参与了氨基酸代谢。在高硼胁迫下从敖汉苜蓿小花中共筛选到97个差异物质,主要参与氨基酸代谢、碳水化合物代谢、脂质代谢、嘌呤代谢和嘧啶代谢;从种子中共筛选并定性到16个差异物质,主要参与氨基酸代谢。5.综合以上分析发现,在硼胁迫下紫花苜蓿小花与种子主要通过调控氨基酸代谢、碳水化合物代谢、脂质代谢和三羧酸循环等代谢途径来适应硼胁迫,从而适应硼营养缺乏或过量环境。
[Abstract]:Boron is one of the essential trace elements of alfalfa. It can promote the normal development of the reproductive organs of alfalfa, be beneficial to the flowering and fruiting, and immeasurable effect on improving the yield and quality of alfalfa seeds. The limit of the amount and excess of alfalfa to boron is very small. The mechanism of boron nutrition in alfalfa and the mechanism of boron stress in the reproductive organs of alfalfa are studied. It is helpful to use boron fertilizer in the actual production process and improve the yield of Alfalfa effectively. On the basis of actual production experiments, the experimental materials of the Aohan alfalfa flower and seed seed are used as the experimental materials, and the proteomics and metabolic groups are applied. To study the changes and metabolic pathways of protein and small molecule metabolites under boron stress and explore the response mechanism of the reproductive organs of Alfalfa in response to boron stress. The main contents are as follows: 1. the application of 0,0.04,0.08,0.12 and 0.16% concentration of boron to the growth of Alfalfa for second years was carried out. The results showed that the application of boron fertilizer was effective. Increase the number of pollen, pollen vigor, seed yield, yield components and seed quality of alfalfa. In general, the effect of 0.08% concentration treatment group is better, and the effect of increasing yield is remarkable. The 0.12% treatment group has the phenomenon of falling flower, which reduces the seed yield. When the spraying concentration reaches 0.16%, it has the toxic effect. So 0% concentration is chosen. For boron deficiency treatment, 0.08% concentration as the optimum fertilizer amount, 0.16% concentration as high boron treatment, the antioxidant enzyme activity, MDA, proline and soluble sugar content of Alfalfa small flowers and seeds treated by.2., 0% (boron deficiency), 0.08% (control) and 0.16% (Gao Peng) boron treated alfalfa were measured. The results showed that the alfalfa under boron deficiency stress The activity of antioxidant enzymes and the content of MDA and proline decreased in different degrees. Under the high boron treatment, the antioxidant enzyme activity and MDA content of alfalfa seeds increased, while the content of proline and soluble sugar decreased. The effect of high boron stress on the alfalfa small flowers was not significant, but the effect of.3. on the seeds of the seeds was a certain effect on the use of 2-DE technology. 0%, 0.08% and 0.16% boron treated alfalfa flowers and seed proteins were separated and analyzed by PDQuest image software, LC-MS/MS mass spectrometry, Mascot software and Uniprot database. Under boron deficiency stress, 12 differentially identified proteins were successfully identified from the alfalfa small flowers, mainly involved in carbohydrate metabolism and lipid. Metabolism. 11 differentially identified proteins were identified from the seeds, mainly involved in carbohydrate metabolism and amino acid metabolism. Under high boron stress, 14 differential proteins were screened from the alfalfa small flower, mainly involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism and secondary metabolism. 11 differential proteins were screened from the seeds. The amino acid metabolism, carbohydrate metabolism and lipid metabolism were involved. Ohan alfalfa protein thioesterase was downregulated under boron deficiency and high boron stress. The NAD dependent aldehyde dehydrogenase family protein up regulated the expression of.NAD (P) combined with Rosman foldable protein in high boron treatment under boron deficiency and high boron stress. Alfalfa was expressed in both small flowers and seeds. It was the key regulatory protein of alfalfa to respond to boron toxicity,.4. using GC-MS technology to analyze the metabolite spectrum of the small flowers and seeds of Alfalfa under boron deficiency and high boron stress. The VIP value of the first principal component of the OPLS-DA model (threshold 1) was combined with the p value of the single dimension statistical (t test) to find the difference. A total of 50 different substances, including carbohydrate metabolism and amino acid metabolism, were mainly involved in the metabolism of carbohydrates and amino acids. 13 different substances were selected from the seeds and mainly involved in the metabolism of amino acids. Under the stress of high boron, 97 different substances were selected from the alfalfa small flower, and they were mainly involved in the amino acid generation. Metabolism of carbohydrates, lipid metabolism, purine metabolism, and pyrimidine metabolism; screening and qualitative analysis from the seeds to 16 different substances, mainly involved in the amino acid metabolism.5. synthesis analysis found that under boron stress, alfalfa small flowers and seeds are mainly regulated by amino acid metabolism, carbohydrate metabolism, lipid metabolism, and three carboxylic acids. Ring and other metabolic pathways are adapted to boron stress, so as to adapt to boron deficiency or excessive environment.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S541.9

【参考文献】