【作者】 张保青；

【导师】 杨丽涛；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 甘蔗(Saccharum officenarum L.)为禾本科甘蔗属植物,属于热带、亚热带作物,其光合效率高,光饱和点高,而二氧化碳补偿点低,光呼吸率低,光合强度大,以蔗茎为收获物,因此生物产量和经济产量高。但甘蔗对自然资源有一定的要求,特别是对温度和水分。在我国,近年来气候变化异常,极端低温频发,在甘蔗的生长后期常遭受自然低温伤害,低温使甘蔗生产和制糖工业生产的发展受严重影响,导致不同程度的经济损失。本研究采用自然低温从生理生化水平对其品种的抗寒性进行比较研究,探讨各指标与抗寒性的关系,运用蛋白质组学方法比较甘蔗在低温胁迫下的蛋白质表达情况,利用同源克隆等技术对一些抗寒基因进行克隆和表达分析,所获主要结果如下：1.在大田自然低温条件下,以21个常规甘蔗品种作为材料,研究不同甘蔗品种在遭受长时间持续低温时的形态和生理生化特性的变化,结果表明：在长时间自然低温胁迫下,甘蔗叶片、生长点、侧芽、蔗茎均受到不同程度伤害；甘蔗在自然降温过程中,不同甘蔗品种的生理指标反应程度各异,叶绿素受寒害后前期降解慢,后期降解逐渐加剧,可溶性糖含量因品种抗寒性不同而表现不一,可溶性蛋白质在低温持续时表现迟钝,脯氨酸和丙二醛(MDA)含量在低温中增加,但因品种抗寒性不同而积累量不同。选择多个生理生化指标,采用模糊隶属函数法,将供试品种抗寒性划分为高抗寒型、抗寒型和低抗寒型3级。2.利用双向电泳技术,选取抗寒性差异大的2个品种桂糖28号和园林6号作为材料,研究低温胁迫对甘蔗生长后期的茎尖蛋白质的差异表达情况。结果表明：经PDQuest软件对蛋白质2-DE图谱进行分析,检测到1000多个有效蛋白点,并找到蛋白质丰度变化在2倍以上的蛋白点45个,部分蛋白点呈现上调表达,而部分蛋白点呈现下调表达,因品种抗性不同而表现各异。推测甘蔗在低温胁迫条件下,蛋白质丰度呈动态性变化。3.运用质谱技术,研究了低温胁迫对甘蔗生长后期茎尖中差异表达的45个蛋白质点,进行了质谱鉴定和生物信息分析,结果表明：45个差异蛋白点经质谱鉴定后,有35个蛋白点被成功鉴定。对所鉴定的蛋白质根据功能分类分为7类。其中参与自由基清除的7个；参与光合作用3个；参与细胞信号转导3个；参与蛋白加工5个；参与细胞生长和分裂2个；参与基础代谢(包括糖代谢、氮代谢、碳代谢、能量代谢、次生代谢以及RNA加工等)11个；未知功能蛋白4个。甘蔗对低温胁迫应答机制是一个涉及多系统、多水平的综合过程。4.采用RT-PCR和同源克隆技术,克隆获得了甘蔗过氧化物酶基因(SoPOD1)、甘蔗20S蛋白酶体α亚基基因(So20S)、甘蔗α-微管蛋白基因(SoTUA)以及5个甘蔗钙调磷酸酶B类似蛋白家族基因CSoCBL1, SoCBL3, SoCBL5, SoCBL6和SoCBL9)的cDNA全长。利用qRT-PCR技术研究了上述基因在低温胁迫下在不同抗性甘蔗品种中的的动态表达情况。在低温胁迫下,两个甘蔗品种SoPODl基因表达先升高后降低,但抗寒强的GT28品种SoPOD1基因表达量上升速度要比不抗寒品种YL6快；So20S基因的表达在两个甘蔗品种中的变化不一,在GT28中其变化趋势为先升后降,在YL6中其变化趋势为缓慢升高；SoTUA在两个品种中的变化总体上趋势一致,但在含量上,GT28中的表达量明显要高于在YL6中的；SoCBL1和SoCBL9基因的表达水平在两个品种中是基本一致；SoCBL3在抗寒的GT28品种中表达量出现明显下降,在YL6品种则呈先上升后急剧下降趋势；SoCBL5的表达水平在两个品种总体出现下降趋势,而SoCBL6基因在GT28和YL6品种在低温胁迫下的表达趋势为上升,即使是继续低温,基因的表达水平有所下降,但仍高于对照。CBL家族成员中起作用的关键基因有可能是SoCBL6和SoCBL5。以上基因都在甘蔗低温胁迫下有一定的应答反应,其基因的功能不同,所在低温胁迫过程中参与的防御途径也不相同,为加强甘蔗抵御低温胁迫及其它非生物胁迫提供了重要的理论基础。更多还原

【Abstract】 Sugarcane (Saccharum officenarum L.) belongs to gramineous genus, originated from tropical and subtropical areas, which has high photosynthetic efficiency, high light saturation point while low co2compensation point, low photorespiration rate and high photosynthetic intensity, and its harvest organ is stalk, so sugarcane crop has high biomass yield and economic productivity. But it has certain requirement for natural resources, especially for temperature and moisture. In China, different degrees of economic losses have been recorded every year in recent years due to abnormal climate damage, especially frequent occurrences of extreme low temperature during late growth stage, which have severely hampered the sustainable development of sugar cane and sugar industry. This study investigated the cold tolerance of different sugarcane varieties and the relationshiop between the cold resistance and physiological and biochemical characteristics, and two typical sugarcane varieties with different cold resistance were selected for proteome comparison, and some differentially expressed cold resistance genes were cloned by using homology-based cloning techniques. The main results obtained were as follows:1. Under natural low temperature conditions in field,21sugarcane varieties were used as materials to study the changes of morphological physiological and biochemical characters. The results showed that the leaves, growing point, lateral bud and cane of sugarcane were damaged in different degrees under long duration of naturally decreasing low temperature stress. The physiological parameters in different sugarcane varieties are different during the natural cooling process. Chlorophyll was found to be decomposed slowly at early stage, and faster later. Soluble sugar content was different with different cold hardiness of sugarcane varieties, which showed insensitive under continous low temperature stress. The content of proline and malondialdehyde (MDA) increased during low temperature, but the accumulation was different in the sugarcane varieties with different hardiness. Based on multiple physiological and biochemical parameters, subordinate function was employed to evaluated the cold resistance of the21sugarcane varieties, and they were divided into3levels.2. By using2-DE technology, two sugarcane varieties, GT28with high cold resistance and YL6with low cold resistance, were selected as materials to study the differential expressions of stem tip proteins under stress at the late growth stage. The results showed that more than1000effective protein spots were detected by analyzing the2-DE mapping with PDQuest software, and45protein spots were found changed more than2times in protein abundance. Parts of the differential protein spots were up-regulated while others were down-regulated, depending on variety resistance. It is speculated that sugarcane protein abundance showed dynamic change under low temperature stress.3. The45differentially expressed protein spots in sugarcane stem tip at late growth stage were identified with mass spectrometry, and analyzed with bioinformatics. The results showed that35protein spots were identified successfully. The identified proteins were divided into seven categories according to their functions. And7of them involved in the free radical scavenging,3involved in photosynthesis,3participated in signal transduction,5participated in protein processing,2involved in cell growth and division,11participated in the basal metabolisms including carbon metabolism, nitrogen metabolism, sugar metabolism, energy metabolism, secondary metabolism and RNA processing, etc., and4remained unknown in their functions. The mechanism of sugarcane responses to low-temperature stress is an integrated process which involve multi-systems and multi-levels.4. The cDNA full-length of the genes SoPOD1, So20S, SoTUA, and5SoCBL family genes SoCBL1, SoCBL3, SoCBL5, SoCBL6and SoCBL9was cloned by RT-PCR and RACE techniques. The dynamic expressions of these genes in different resistant sugarcane varieties under cold stress were studied with qRT-PCR techniques. Under cold stress, the expression levels of SoPODl gene in two sugarcane varieties increased at first and then decreased, but that in the strong cold resistance variety GT28rose faster than that the cold sensitive variety YL6. The expression of So20S gene was different between the two varieties, which showed increasing at first and then decreasing while it showed slowly increasing in YL6.The expression tendency of SoTUA gene in two sugarcane varieties was the same, but the content was significantly higher in GT28than in YL6. The expressions of SoCBLl gene and SoCBL9gene were basically identical. The expression of SoCBL3gene showed significant decrease in GT28, but firstly increase and then sharp decrease in YL6. SoCBL5gene showed an overall declining trend in two sugarcane varieties. The expression trend of SoCBL6gene was increasing in both varieties GT28and YL6dunder cold stress, and it always higher than the control even when the expression level showed slight decrease after continuous cold stress. It was likely that the key genes in CBL family members were the genes SoCBL6and SoCBL5. All these genes in sugarcane had certain responses to cold stress, but the functions of these genes were different, and the defense pathways they involved were also different under the process of cold stress, which provide important molecular basis to strengthen sugarcane against cold stress and other abiotic stress.更多还原