玉米苗期抗冻生理响应及其转录组调控分析

发布时间：2018-05-08 22:37

本文选题：玉米 + 冻害　；参考：《东北农业大学》2017年博士论文

【摘要】：随着温室气体排放增加,全球气候变暖导致极端天气事件发生频率明显增加,各种极端气候事件,如强寒潮、干旱和洪涝灾害等对农业生产造成了严重影响。玉米是世界上最重要的粮食作物、饲用作物和能源作物之一。而且由于起源于热带地区,玉米是典型喜温喜光的C4植物,对温度条件要求较高,易受低温冷(冻)害的影响。在我国玉米生产中,春季和秋季的霜冻对玉米产量易造成严重影响,尤其是对北方春播玉米区和西北灌溉玉米区,而且春季霜冻的频次要高于秋季霜冻。东北地区是我国玉米的主产区,而黑龙江省玉米常年种植面积已达到粮食作物总面积的一半以上,为我国玉米播种面积最大的省份。近年来由于玉米需求的刚性增长和玉米种植比较效益的提高,黑龙江省玉米种植区域已经发展到第6积温带,玉米跨区种植的情况非常严重。与此同时,近年来黑龙江省春季常有雨雪冰冻天气,玉米播种期易遇倒春寒而推迟,直接影响玉米的正常播种,而且玉米幼苗期也易遭受倒春寒而出现冻害损伤,影响玉米的正常生长,并最终造成减产。因此,开展玉米苗期抗冻性研究,筛选玉米抗冻种质资源,探究玉米苗期抗冻性生理生化机制,挖掘玉米在低温胁迫下的抗冻基因,进而开展玉米抗冻性分子标记辅助育种等相关方面的研究,对于解决黑龙江省玉米冷(冻)害问题具有重要而深远的理论和实践意义。本研究利用30份黑龙江省常用玉米自交系,开展了玉米苗期抗冻性鉴定及相关生理指标测定,同时结合转录组测序技术,对具有不同抗冻性的玉米自交系苗期冻害胁迫响应基因及相关代谢通路进行了比较分析,研究结果如下:一、玉米苗期抗冻性的高效鉴定方法本研究选取三叶一心期的玉米自交系幼苗放置于-1℃的培养箱中分别进行1h、3h和5h的冻害处理,之后放置于正常培养条件的培养箱中恢复3天,3天之后统计幼苗存活率,以幼苗存活率来作为抗冻性的测量指标。结果显示冻害胁迫处理3小时的幼苗存活率从5.01%到97.3%不等,呈现正态分布,能够有效的区分不同自交系的抗冻性,因此将冻害胁迫时间3小时作为有效鉴定玉米苗期自交系抗冻性的处理时间。并且筛选出两份极端自交系用于后续研究,其中,自交系KR701在冻害处理3小时之后的幼苗存活率为97.3%,作为抗冻自交系;Hei8834在冻害处理3小时之后的幼苗存活率为5.01%,作为冻害敏感自交系。二、冻害胁迫下玉米苗期生理机制分析Hei8834在冻害处理之后表现出明显的叶片萎蔫、失绿,台盼蓝染色后显微观察也发现冻害处理之后Hei8834的叶片气孔张开并呈现深蓝色,说明其叶片受冻害损伤严重。同时,冻害处理后,Hei8834的相对电导率在0.5小时和1小时分别为0.13和0.87,表明Hei8834在冻害处理0.5小时到1小时之间冻害损伤明显加剧;同时超氧化物歧化酶活性在冻害处理后1小时达到最高值57.3 U/mg,之后便开始下降;过氧化物酶活性在冻害处理后0.5小时达到最大值53.7 U/mg,之后也呈现下降趋势;可溶性蛋白和可溶性糖含量都是在冻害处理之后一直缓慢升高。KR701的相对电导率在冻害处理之后一直维持0.2以下;超氧化物歧化酶活性在冻害处理之后上升趋势高于Hei8834,而且在2小时达到最高值80.8,之后略有降低;过氧化物酶活性在冻害处理之后一直处于上升趋势;可溶性蛋白含量在冻害处理之后也是上升趋势;可溶性糖含量在冻害处理之后也是一直升高,直到2小时之后维持相对稳定水平,并有下降趋势。因此,冻害敏感自交系Hei8834在冻害处理1小时之后,冻害损伤明显,冻害抵抗机制已失效;而抗冻自交系KR701在冻害处理3小时以内,冻害损伤较小,冻害抵抗机制有效的保护了细胞免于冻害损伤,但是在2小时到3小时之间,有些生理指标(超氧化物歧化酶活性和可溶性糖)已经开始略微呈现下降趋势,暗示冻害处理时间如再延长,抗冻自交系KR701的抗冻机制也不能有效抵抗冻害胁迫。三、不同抗冻性玉米自交系幼苗冻害胁迫前后的转录组测序分析对这两份极端材料在幼苗期冻害处理之前和冻害处理之后进行转录组测序,共得到大约154.4百万长度为101 bp的序列,其中78.84%(121.8百万)的序列能比对在玉米参考基因组Zea_mays_Ensembl_AGPv3。聚类分析及主成分分析(PCA)结果显示,抗冻自交系KR701在冻害处理之前和冻害处理之后的样本归为一类,冻害敏感自交系Hei8834在冻害处理之前和冻害处理之后的样本归为一类,导致不同样本间基因表达量最大差别的因素为自交系之间的差异。4个不同处理的7个样本中,共有19794个基因表达,其中有360个基因在抗冻自交系KR701中经过冻害胁迫之后特异表达,489个基因在冻害敏感自交系(Hei8834)在冻害胁迫之后特异表达。共有4550个基因在4个不同的处理(两份自交系之间以及冻害处理前后)中的表达量存在差异(即为差异表达基因,DEG)。对这些特异表达基因和差异表达基因分别进行了基因本体论分析(GO分析),结果发现冻害处理后冻害敏感自交系特异表达的基因与抗冻自交系特异表达的基因相比,特异的参与了一些生物学过程,如低温响应、有机酸转运,此外泛素连接酶复合体组分也显著富集。这也从转录水平上说明冻害敏感自交系在冻害处理中受到的损伤较为严重,从而触发了更多的生物学过程。同时,虽然许多GO条目在两个自交系冻害处理后特异表达的基因中都有出现,但是这些GO条目在两个自交系所富集的显著程度不同,说明参与同一GO的基因在两个自交系中所占的比例是不同的。对抗冻自交系冻害处理前后的439个差异基因和冻害敏感自交系冻害处理前后的852个差异基因分别进行了GO分析,结果发现在生物学过程分类中,抗冻自交系中富集最为显著的是冻害响应,而冻害敏感自交系富集最为显著的是低温响应;在分子功能分类中,抗冻自交系中富集最为显著的是结合活性,而冻害敏感自交系富集最为显著的是蛋白质酪氨酸激酶活性;在细胞组分分类中,抗冻自交系中有三个GO条目显著富集,均为膜组分相关,而冻害敏感自交系富集最为显著的是泛素连接酶复合体。此外我们对在两个自交系间冻害响应存在显著差异的基因进行了拟南芥同源基因功能检索,结果鉴定到许多参与逆境响应和植物激素信号通路相关的基因。这些基因的后续分析将会增加我们对玉米幼苗在早期冻害胁迫下响应冻害的分子机制的认识,并对玉米抗低温育种提供良好的育种策略。最后,挑选出30个基因进行实时荧光定量PCR(qRT-PCR)验证,结果显示转录组测序结果和qRT-PCR验证结果在抗冻材料和冻害敏感材料中的相关系数分别是80%和50%,说明转录组分析结果与qRT-PCR结果吻合度较高。
[Abstract]:With the increase of greenhouse gas emissions, global warming has led to an obvious increase in the frequency of extreme weather events, and a variety of extreme weather events, such as strong cold tide, drought and flood, have seriously affected agricultural production. Corn is one of the most important food crops, forage crops and energy crops in the world. In the region, maize is a typical warm and warm C4 plant with high temperature and cold (freezing) damage. In maize production in China, spring and autumn frost have a serious effect on maize yield, especially in northern spring sowing corn and northwest irrigated corn region, and the frequency of frost in spring is higher than that in autumn frost. The northeast region is the main producing area of Maize in China, and the annual planting area of Maize in Heilongjiang has reached more than half of the total area of grain crops, which is the largest province in China. In recent years, because of the rigid growth of corn demand and the improvement of the comparative benefit of Maize planting in recent years, the maize planting area in Heilongjiang has developed to the first. In the 6 product of temperate zone, the situation of Maize Cross planting is very serious. At the same time, in recent years, the spring of Heilongjiang often has the freezing weather of rain and snow, the corn sowing period is easy to meet the spring cold and postpone, directly affects the normal sowing of corn, and the maize seedling stage is also vulnerable to the cold injury, which affects the normal growth of corn, and eventually causes the corn. Therefore, to carry out the research on the frost resistance of Maize at the seedling stage, select the germplasm resources of the corn frost resistance, explore the physiological and biochemical mechanism of the cold resistance of Maize at the seedling stage, excavate the antifreeze gene of corn under the low temperature stress, and then carry out the research on the related aspects of the corn frost resistance molecular marker assisted breeding, which can solve the problem of cold (freezing) damage to Maize in Heilongjiang province. There are important and far-reaching theoretical and practical significance. In this study, 30 common maize inbred lines in Heilongjiang province were used to identify the resistance of Maize at seedling stage and to determine the related physiological indexes. At the same time, the response genes and related metabolic pathways of the cold damage stress of maize inbred lines with different frost resistance were carried out in combination with the sequencing of the transcriptional sequence. The results are as follows: 1. High efficiency identification method for the frost resistance of Maize at the seedling stage. This study selected the maize inbred lines of the three leaves and one heart stage to carry out the freezing injury treatment of 1H, 3H and 5h respectively in the -1 C incubator. Then the seedlings were placed in the normal culture incubator for 3 days, and the seedling survival rate was counted after 3 days, and the seedlings were counted. The survival rate was used as an indicator of frost resistance. The results showed that the survival rate of the seedlings for 3 hours was from 5.01% to 97.3%, showing a normal distribution, which could effectively distinguish the frost resistance of different inbred lines. So 3 hours of freezing stress time was used as an effective method to identify the freezing resistance of maize inbred lines. Two extreme self inbred lines were used for follow-up studies. Among them, the survival rate of the seedlings of the inbred line KR701 after 3 hours of freezing injury was 97.3%, as a freeze-resistant inbred line. The survival rate of the seedlings after 3 hours of freezing injury treatment was 5.01%, as a freezing sensitive inbred line. Two, the physiological mechanism of Maize Seedling under the frost stress was analyzed by Hei8834 in the frost injury. After the treatment, the leaf blowhole of Hei8834 was open and dark blue after the freezing injury treatment, indicating that the blade was damaged by frost damage. At the same time, the relative conductivity of Hei8834 was 0.13 and 0.87, respectively, when the freezing injury treatment was 0.5 and 1 hours, indicating that Hei8834 was frozen. At the same time, the activity of superoxide dismutase (SOD) reached a maximum value of 57.3 U/mg at 1 hours after the treatment of freezing injury, and then began to decrease, and the peroxidase activity reached the maximum value of 53.7 U/mg at 0.5 hours after the freezing injury treatment, and then it also showed a downward trend, and soluble protein and soluble sugar contained the soluble sugar. The relative conductivity of.KR701 has been increased slowly after the freezing injury treatment, and the relative conductivity of the.KR701 has been maintained below 0.2 after the freezing injury treatment, and the activity of superoxide dismutase is higher than that of Hei8834 after freezing injury treatment, and it reaches the maximum value of 80.8 at 2 hours, then decreases slightly, and the activity of peroxidase is always in the cold injury treatment. The content of soluble protein was also rising after freezing injury treatment, and the content of soluble sugar increased after freezing injury treatment, and maintained relative stability level until 2 hours after 2 hours. Therefore, the frost damage sensitive self inbred line was obviously damaged after 1 hours of freezing injury treatment, and the mechanism of freezing injury resistance was already found. While the freezing damage treatment was less than 3 hours, the frost damage was less than 3 hours, and the freezing injury resistance mechanism effectively protected the cell from freezing damage, but between 2 hours and 3 hours, some physiological indexes (superoxide dismutase activity and soluble sugar) had begun to decrease slightly, suggesting the freezing injury treatment time, such as The frost resistance mechanism of the anti freeze inbred line KR701 could not effectively resist the frost damage. Three, the transcriptional sequence analysis of the different frost resistance maize inbred lines before and after the freezing stress of the two extreme materials was sequenced before the freezing injury treatment and the freeze injury of the seedling stage, and a total of about 154 million 400 thousand of the length of 101 BP was obtained. The sequence of 78.84% (121 million 800 thousand) was compared with the results of Zea_mays_Ensembl_AGPv3. cluster analysis and principal component analysis (PCA) in maize reference genome. The results showed that the samples of the antifreeze KR701 inbred line before the freezing injury treatment and the freezing injury treatment were classified as a class. The freezing injury sensitized inbred line Hei8834 was before the freezing injury treatment and the freezing injury treatment. The following samples are classified into one class. The factors that lead to the maximum difference in gene expression among different samples are 7 different.4 samples of the inbred lines. There are 19794 genes, of which 360 genes are specifically expressed in the anti freeze inbred line KR701 after freezing stress, and the 489 genes are in the cold sensitive inbred line (Hei8834). The expression of 4550 genes in 4 different treatments (between two inbred lines and before and after freezing injury treatment) were different (i.e., differentially expressed genes, DEG). The gene ontology analysis (GO analysis) of these specific and differentially expressed genes was carried out, and the results of freezing injury treatment were found. The specific genes expressed in the sensitive inbred lines of the post freeze injury are specifically involved in some biological processes, such as the low temperature response, the organic acid transport, and the ubiquitin ligase complex components, as well as the damage caused by the freezing injury sensitive inbred lines in the freezing injury treatment. More serious and more biological processes were triggered. While many of the GO entries appeared in the two inbred line specific genes after freezing injury treatment, these GO entries were significantly different in the two inbred lines, indicating that the proportion of the genes involved in the same GO is different in the two inbred lines. 852 different genes before and after freezing injury treatment before and after freezing injury treatment of frozen self inbred lines were analyzed by GO. The results showed that in the biological process classification, the most significant accumulation of freezing injury in the antifreeze inbred lines was the freezing injury response, and the most significant accumulation of cold damage sensitive inbred lines was the low temperature response. In the molecular functional classification, the most significant accumulation of the antifreeze inbred lines is the binding activity, while the most significant enrichment of the freezing sensitive inbred lines is the protein tyrosine kinase activity. In the cell classification, three GO entries are significantly enriched in the antifreeze inbred lines, all of which are related to the membrane components, and the most significant enrichment of the freezing sensitive inbred lines is the most significant. It is the ubiquitin ligase complex. In addition, we searched the Arabidopsis homologous gene for genes of Arabidopsis homologous genes with significant differences in the freezing damage response between two inbred lines. The results identified many genes involved in stress response and plant hormone signaling pathways. Subsequent analysis of these genes will increase our early growth in maize seedlings. A good breeding strategy was provided for maize breeding in response to cold damage under the cold damage stress. Finally, 30 genes were selected for real-time fluorescence quantitative PCR (qRT-PCR) verification. The results showed that the correlation coefficient of the transcriptional sequence and qRT-PCR verification results in the antifreeze material and the freezing sensitive material was 8, respectively. 0% and 50%, indicating that the transcriptome analysis results are in good agreement with the qRT-PCR results.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513;S42

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