【作者】 闫锋；

【导师】 庞保平； 娄永根；

【作者基本信息】 内蒙古农业大学 ， 作物栽培与耕作学， 2009， 博士

【摘要】 植食性昆虫取食植物后,使受害植物释放一些在质和(或)量上与健康植株不同的挥发性化合物,这些化合物会对昆虫自身、其它植食性昆虫等产生影响。另外,植物细胞能通过类受体蛋白激酶感受生物和非生物因子的刺激,并通过细胞膜将胞外的信号传导到胞内,从而激活胞内下游的防御基因,使植物具有防御功能。本研究的主要结果如下:1.利用顶空动态吸附法对健康稻株、二化螟和稻纵卷叶螟取食为害24小时的水稻挥发物进行捕集。结果表明,在2种虫害苗和健康苗中,共捕集到28种挥发物组分,其中定性的16种,包括萜类化合物9种,烷烃类化合物4种,酮类、醇类、酯类化合物各1种。从健康稻株中共捕集到22种挥发物,已鉴定出的化合物有2-庚酮、α-蒎烯、柠檬烯、芳樟醇、α-古巴烯、正十四烷、姜烯、α-雪松烯、雪松醇、正十七烷、正十八烷和正十九烷共12种化合物。在二化螟幼虫取食诱导的水稻挥发物中共捕集到26种挥发物,鉴定出的化合物有α-蒎烯,柠檬烯,α-古巴烯,α-雪松烯,芳樟醇氧化物,芳樟醇,雪松烯和姜烯等萜类化合物8种,其他有2-庚醇,2-庚酮,正十七烷,正十八烷,正十九烷和正十四烷。在稻纵卷叶螟幼虫取食为害苗中共捕集到26种挥发物,其中鉴定的化合物有萜类化合物9种,分别为α-蒎烯,柠檬烯,α-古巴烯,β-石竹烯,α-雪松烯,芳樟醇氧化物,芳樟醇,雪松醇和姜烯;其它化合物7种,分别为2-庚酮,2-庚醇,水杨酸甲酯,正十七烷,正十八烷,正十九烷和正十四烷。2.利用EAG技术测定了二化螟雄虫和稻纵卷叶螟雌、雄虫对水稻挥发物的电生理反应。结果表明,二化螟雄虫除正十四烷以外,对2-庚醇、正十八烷、正十九烷、柠檬烯、芳樟醇和2-庚酮6种化合物反应值均随样品浓度的增加而增大。二化螟雄虫对上述7种化合物的阈值为1ng/μl。稻纵卷叶螟雌蛾除正十八烷和正十九烷外,其对2-庚醇、正十四烷、柠檬烯、芳樟醇和2-庚酮5种化合物反应值均随样品浓度的增加而增大。稻纵卷叶螟雄蛾除正十八烷和柠檬烯外,其对2-庚醇、正十四烷、芳樟醇和2-庚酮4种化合物反应值均随样品浓度的增加而增大。稻纵卷叶螟雌、雄蛾的EAG反应值比较发现,除芳樟醇和正十八烷对其雌、雄蛾触角EAG反应值差异不显著,2-庚醇、正十四烷、2-庚酮和柠檬烯均使雌蛾的EAG值发生值显著比雄蛾大。尤其对柠檬烯的浓度剂量表现更为明显,当柠檬烯浓度低于0.001ng/μl时,对雄虫影响较大,当浓度大于0.1ng/μl时反而对其雌虫影响较大。3.二化螟取食为害稻株对灰飞虱生长发育的影响。田间调查发现二化螟为害苗上的灰飞虱数量极显著地比健康苗上的灰飞虱数量多。室内研究表明,二化螟为害稻株上的灰飞虱若虫的生长历期显著比健康稻株上的生长历期短,同时雌成虫寿命显著比雄虫寿命长,对翅型分化、性比及产卵前期无显著影响,而为害苗上灰飞虱的产卵量显著比健康苗上的多。在受害株上的灰飞虱若虫最喜食二化螟的取食部位的茎秆。雌成虫选择性试验表明,灰飞虱比较显著偏向受害株。从二化螟为害株和健康株中共检测到17种游离氨基酸,比较二化螟为害9天的稻株与健康稻株中游离氨基酸,发现苏氨酸含量在受害株中有所下降,而天冬氨酸、丝氨酸、甘氨酸、异亮氨酸、亮氨酸和苯丙氨酸的含量在受害株中显著增多,谷氨酸、丙氨酸、半胱氨酸、缬氨酸、甲硫氨酸、酪氨酸、赖氨酸、组氨酸、精氨酸和脯氨酸含量变化不显著。4.通过RT-PCR的方法,从二化螟取食后的水稻中分离出OsRLK基因,经测序和Blastn、Blastp比较分析,确定所克隆的基因为OsRLK。将该基因和表达载体连接后经一系列连接转化后,获得了过量表达载体,利用农杆菌介导法将该基因成功转入到水稻中,并获得了含有OsRLK的过量表达转基因植株。更多还原

【Abstract】 After being fed by phytophagous insects, the damaged plants can produce and release the volatile compounds which are different qualitatively or/and quantatively from the healthy plants, and the compounds can affect the phytophagous insects themselves and others. In addition, plant cells can feel the stimulation of the biotic and abiotic factors through receptor-like protein kinase and conduct the signal from extracellular to intracellular through the cellar membrane, which can activate the defense genes of intracellular downstream and make the plants get the defense function. The main results as following:1.Using the dynamic techniques for headspace desorption, the rice volatiles from the healthy plants and damaged plants were trapped after damaged by Chilo suppressalis and Cnaphalocrocis medinalis for 24h. The results showed that 28 kinds of volatiles compounds were trapped from the damaged and healthy seedlings, and 16 kinds of volatile compounds were qualitatively determined including 9 terpenoid, 4 alkanes, 1 ketones, 1 alcohols and 1 esters. 22 volatiles were trapped from the healthy seedlings, and 2-heptanone,α-pinene, (+)-limonene, linalool,α-copaene, n-tetradecane, zingiberene, (-)-α-cedrene, (+)-cedrol, n-heptadecane, n-octadecane and n-nonadecane were identified. 26 volatiles were trapped from the rice damaged by C. suppressalisr larvae, and terpenoidα-pinene, (+)-limonene,α-copaene, (-)-α-cedrene, (E)-linalool oxide, linalool, (+)-cedrol,zingiberene, 2-heptanone, 2-heptanonol, n-heptadecane, n-octadecane, n-nonadecane and n-tetradecane were identificated. 26 volatiles were trapped from the seedlings damaged by C. medinalis larvae, and 9 terpenoid, includingα-pinene, (+)-limonene,α-copaene,β-caryophyllene, (-)-α-cedrene, (E)-linalool oxide, linalool, (+)-cedrol and zingiberene, were identificated, and the others (2-heptanone, 2-heptanonol, MeSA, n-heptadecane, n-octadecane, n-nonadecane and n-tetradecane) were also identificated.2.Using EAG technology, the response of C. suppressalis male adults, and C. medinalis male and female adults to the rice volatiles from the rice seedlings was tested. The results showed that Except n-tetradecane, the response value of C. suppressalis male adult to 2-heptanonol, n-octadecane, n-nonadecane, limonene, linalool and 2-heptanone increased with increasing sample concentration. The threshold of C. suppressalis male adult to seven substance above is 1ng/μl. Except n-octadecane and n-nonadecane, the response value of C. medinalis female adult to 2-heptanonol, n-tetradecane, limonene, linalool and 2-heptanone increased with increasing sample concentration. Except n-octadecane and limonene, the response value of C. medinalis male adult to 2-heptanonol, n-tetradecane, linalool and 2-heptanone increased with increasing sample concentration. Comparing the response value of EAG between C. medinalis female adult and male adult, it was found that the EAG response value of linalool and n-octadecane to the female and male adult had no obvious difference, and 2-heptanonol, n-tetradecane,2-heptanone and limonene cause the EAG response value of female adult significantly greater than that of male adult. Especially the dose of limonene concentration, when the limonene concentration was lower than 0.001ng/μl, it had great influence on male adult. But when limonene concentration was higher than 0.001ng/μl, it had great influence on female adult.3.Field investigation showed that the number of Laodelphax striatellus on the seedlings damaged by C. suppressalis was much more than that on the healthy seedlings. The growth period of L. striatellus nymph on the seedlings damaged by C. suppressalis was much shorter than that on the healthy seedlings, and the life of female adults was further longer than that of male adults, but the wing dimorphism, sex ratio and preoviposition period had no difference whereas the fecundity of L. striatellus on the damaged seedlings was much more than that on the healthy seedlings. L. striatellus nymphs on the damaged plants preferred to the stem of feeding sites of C. suppressalis. L. striatellus female adults prefered to the damaged plants. 17 free amino acids in the plants damaged by C. suppressalis and healthy plants were detected. Through the comparison of the plants damaged by C. suppressalis and the healthy plants, threonine content in damaged plants decreased somewhat, and the contents of aspartate, serine, glycine, isoleucine, leucine and phenylalanine in damaged plants significantly increased whereas the contents of glutamate, alanine, cysteine, valine, methionine, tyrosine, lysine, histidine, arginine and proline did not significantly change.4.Using RT-PCR, the OsRLK(receptor-like protein kinase)gene from the rice fed by C. suppressalis was isolated, and sequencing and comparative analysis of Blastn and Blastp confirmed that the cloned gene was OsRLK. By a series of connection and transformation after connecting the OsRLK with expression vector, the over expression vector was obtained. The OsRLK was transferred to rice successfully using agrobacterium-mediated transformation and the over expression of transgenic plant including the OsRLK was obtained.更多还原