【作者】 李慧；

【导师】 徐彬；

【作者基本信息】 南京农业大学 ， 草学， 2018， 硕士

【摘要】 柳枝稷(Panicum virgatum)是禾本科黍属多年生暖季型草。其植株高大、生长迅速、根系发达、抗逆性和适应性强,是重要的能源植物和水土保持植物。挖掘柳枝稷的抗逆调控基因并应用于柳枝稷及相关禾本科草的分子育种,进一步提高其抗逆性,对边际土地的修复、改良和利用具有重要意义。植物水通道蛋白在维持植物体内水分状态中扮演重要角色,PIPs是水通道蛋白家族中的一类亚族,能够影响植物摄取水分以及小分子物质的能力。部分PIP亚家族基因可以调控植物的非生物胁迫,尤其是对植物耐旱调控具有有重要的作用。本实验克隆到一个柳枝稷PIP家族基因(PvPIP2;9)基因,并对其功能进行分析,发现在柳枝稷中过量表达了PvPIP2;9基因可以显著提高柳枝稷的耐旱性。主要实验结果如下:(1)系统进化树分析结果表明PvPIP2;9与OsPIP2;4、OsPIP2;7、ZmPIP2;7的同源关系较近;然后用PvPIP2;9与OsPIP2;4、OsPIP2;7、ZmPIP2;7的蛋白序列进行多重序列比对,结果显示它们均具有水通道蛋白典型结构域:6个跨膜结构域和2个保守的NPA结构域。(2)对PvPIP2;9基因的表达模式进行了分析。PvPIP2;9的基因表达受光周期的调控,并呈现节律变化,在光照后表达量升高,光照4小时后达到最高值,而后下降。在冷和干旱处理条件下,PvPIP2;9基因的表达量呈现上调表达,而盐处理和ABA处理条件下,PvPIP2;9基因的表达量呈现下调表达。(3)利用农杆菌介导法遗传转化柳枝稷,获得过量表达PvPIP2;9基因的转基因柳枝稷。对转基因柳枝稷与野生型柳枝稷进行干旱处理,发现转基因柳枝稷的耐旱性显著强于野生型柳枝稷。对植物耐旱性的相关生理指标进行测定,结果表明转基因柳枝稷的电导率显著低于野生型柳枝稷,而叶片相对含水量显著高于野生型柳枝稷,表明转基因柳枝稷能够维持较好的细胞膜完整性,从而使植物能够保持较高的水分含量,从而来应对干旱胁迫;进一步对光化学效率和叶绿素含量进行测定,发现转基因柳枝稷的光化学效率和叶绿素含量显著的高于野生型柳枝稷,说明转基因柳枝稷能够维持较好的光合作用来维持植物正常生长;对水分利用效率进行了测定,发现转基因柳枝稷的水分利用效率显著的高于野生型柳枝稷。综上,PvPIP2;9基因的表达受光照节律影响,并受到干旱等逆境胁迫的影响;在柳枝稷中过量表达PvPIP2;9基因,提高了柳枝稷在干旱条件下的水分利用效率和光合作用并缓解了细胞膜破损,进而增强了柳枝稷的耐旱性。更多还原

【Abstract】 Switchgrass(Panicum virgatum)is a perennial warm-season grass in the grass family(Poaceae).Switchgrass is featured with its tall statue,rapid growth,robust root system,and high resistance to most stresses and adaptive to adverse environment.It has been used as the non-grain-based bioenergy plant as well as the soil and water conservation plant.Identification the resistance genes and applying such knowledge onto molecular breeding of switchgrass and the other related Poaceae species is a promising way to further improve its stress resistance for the purpose of restoration,improvement and utilization of marginal land.Plant aquaporins play an important role in maintaining the water status in plants.PIP is one subfamily of the aquaporins.The PIP proteins affect the ability of plants to take up water and some other small molecules.A number of the PIP subfamily genes can positively regulate plant abiotic stress,especially the adaptation to drought stress.In this study,we cloned and analyzed a switchgrass PIP gene,PvPIP2;9,gene and found that over-expressing the PvPIP2;9 gene significantly improved the drought resistance of switchgrass.The results of this study are as follows:(1)Phylogenetic tree analysis showed that PvPIP2;9 is close to OsPIP2;4,OsPIP2;7,and ZmPIP2;7 in rice and maize.The result of multiple sequence alignment show that PvPIP2;9 as well as its orthologous proteins had the typical 6 transmembrane domains and two conservative NPA motifs in the aquaporin family.(2)The expression pattern of PvPIP2;9 was analyzed.The results showed that PvPIP2;9 was regulated by the circadian rhythm and its expression fluctuated along with the photopeirod.The expression level increased after illumination and reached its maximum after 4 hours,and then decreased.The expression of PvPIP2;9 was also up-regulated by cold and drought treatment,but down-regulated with salt and ABA treatment.(3)Using Agrobacterium-mediated genetic transformation,we generated PvPIP2;9 overexpression transgenic switchgrass lines.The transgenic and wild-type switchgrass were compared for their drought tolerance.We found that the drought tolerance of the transgenic switchgrass was significantly stronger than that of wild-type switchgrass.The related physiological indexes of plant drought tolerance were measured.During the water withholding period,the EL of transgenic switchgrass became significantly lower than that of the wild-type,while the leaf RWC of transgenic switchgrass was significantly higher that of the wild-type,these show that transgenic switchgrass could maintain better cell membrane integrity and higher moisture content to deal with drought stress.We also found that the transgenic switchgrass maintained higher Fv/Fm and chlorophyll contents than those of the wild-type,indicating transgenic switchgrass could maintain better photosynthesis to maintain normal development of plants under the drought stress conditions.Furthermore,we also found that the WUE of transgenic switchgrass were significantly higher than that of wild-type.In conclusion:the expression of PvPIP2;9 gene was affected by the circadian rhythm,and was affected by different abiotic stress factors.Overexpression of PvPIP2;9 gene in switchgrass can improved leaf WUE and photosynthesis under drought treatment and alleviated the damage of cell membrane,thus enhancing the drought tolerance of switchgrass.更多还原