高温对抗性和敏感小菜蛾caspase基因和hsp70基因热激表达及抗性进化的影响
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摘要
小菜蛾是世界性十字花科蔬菜的重大害虫,根据本实验前人研究结果,高温下抗性和敏感小菜蛾生理适合度显著降低,本文选择了高温下显著上调表达的两个基因——caspase和hsp70作为生理适合度指标,通过生化、分子生物学及抗药性测定技术进行了高温对抗性和敏感小菜蛾caspase和hsp70基因热激表达及抗性水平的比较研究。研究揭示了高温条件下小菜蛾种群抗药性水平急剧下降,且这种抗性水平的下降是可遗传的。
caspase是细胞凋亡过程中的关键酶,起到执行细胞凋亡的功能,本文通过同源克隆的策略和RACE技术,克隆得到一个1172bp长的小菜蛾caspase基因(Px-caspase-1),具有完整的开放阅读框,开放阅读框长903bp,编码具有300个氨基酸的caspase蛋白,分子量为33.6kDa。与其它昆虫caspase基因的氨基酸序列比对结果显示,Px-caspase-1)属于效应caspase。高温(32.1、36、40、42、45、47℃)和常温(25℃)预处理对caspase-9(起始caspase)和caspase-3(效应caspase)的酶活力及1px-caspase-1mRNA表达量变化影响的研究结果表明,常温下小菜蛾成虫体内存在caspase-9和caspase-3的酶活性及Px-caspase-1基因表达,高温能够显著诱导caspase-9和caspase-3酶活力增高以及Px-caspase-1mRNA表达上调,且随着温度的升高上调表达的高峰提前出现。此外,高温可导致小菜蛾成虫的死亡率显著增高。上述结果表明,常温25-C下细胞凋亡是小菜蛾的一种正常生命现象,而高温能够显著诱导小菜蛾发生细胞凋亡,使得细胞凋亡基因Px-caspase-1的上调表达高峰提前出现,加速小菜蛾的死亡。比较高温(32.1、40、42℃)处理不同时间敏感和抗性小菜蛾Px-caspase-1基因表达的差异,结果表明抗性小菜蛾Px-caspase-1基因在高温下的热激上调表达显著大于敏感小菜蛾。
在热胁迫下的细胞凋亡与热休克蛋白70家族相关,本文克隆得到小菜蛾hsp70基因(Px-hsp70)序列的全长cDNA。该基因的cDNA序列长2128bp,具有5'非翻译区90bp,3'非翻译区140bp,开放阅读框为1899bp,演绎的蛋白质序列为632aa,分子量为69.3kDa。通过在NCBI上进行的序列比对及功能位点分析,Px-hsp70)禹于hsp70家族成员。通过采用雌、雄幼虫和雌、雄成虫为材料,25、37、42-C预处理3h后,无论在敏感还是抗性种群中整体上,幼虫的Px-hsp70表达量均比成虫Px-hsp70的表达量低,且高温诱导后幼虫Px-hsp70的表达量均无显著变化。在成虫中,高温可显著诱导敏感及抗性小菜蛾Px-hsp70的上调表达,但是高温对敏感小菜蛾Px-hsp70的上调表达程度显著大于对抗性小菜蛾的影响。
这些结果表明,高温可显著抑制敏感和抗性小菜蛾的生理适合度,但是对抗性小菜蛾适合度的抑制作用更为显著。
高温可显著影响小菜蛾的抗药性水平。田间监测的结果表明,夏季7月高温期间小菜蛾抗药性水平急剧下降。小菜蛾在高温下(33.5℃)饲养一代后对阿维菌素抗性水平大幅下降,高温饲养一代后小菜蛾抗药性水平急剧下降可能与高温下抗性小菜蛾适合度显著低于敏感小菜蛾有关。
Plutella xylostella (diamondback moth, DBM) is a destructive cosmopolitan pest of cruciferous crops. As reported, the fitness of the insecticide-resistant (R) and-susceptible (S) DBM was decreased sharply under high temperature. Effects of heat stress on expression of caspase and hsp70genes and level of resistance to insecticide in the R and S DBM were studied by biochemistry, molecular biology and resistant bioassays. The results showed that resistance level of DBM was decreased significantly under heat stress, and this decrease was heritable.
Caspase is the key factor in apoptosis. The caspase gene, named Px-caspase-1, was isolated and sequenced from the DBM. The full-length of Px-caspase-1cDNA was1172bp, including an903bp open reading frame (ORF) that encoded300amino acids. The dedueed protein had a computed molecular mass of33.6kDa. The Px-caspase-1belonged to effector caspase based on its high identity of amino acid sequences to the effector caspase genes from other insect species.
The expression of Px-caspase-1and the enzyme activity of caspase-9and caspase-3under heat stress (32.1、36、40、42、45、47℃) were studied. The results showed that the basic levels of Px-caspase-1expression and caspase-9and caspase-3activities could be detected at25℃. However, the gene expression and the two enzyme activities increased significantly when the adults of DBM were pretreated with high temperature. With the increase of pretreatment temperature and time period, the Px-caspase-1mRNA expression and activities of caspase-9and caspase-3reached the peak value earlier under heat stress than those at25℃. In addition, high temperature could result in the increase of mortality of adult DBM. The results indicated that apoptosis was a normal physiological procession in DBM at normal temperature, and heat stress could significantly induce apoptosis in DBM and cause death of DBM.
The expression of Px-caspase-1between R and S adults DBM under heat stress (32.1、40、42℃) were studied. Significantly increase of Px-caspase-1expression was found in female and male adults of R and S DBM when the adults were pretreated with high temperature, and the extent of up-regulation of Px-caspase-Ⅰ expression in R DBM was significantly higher than in S DBM.
The heat shock protein70family were invoved in apoptosis under heat stress. The heat shock protein gene, named Px-hsp70, was isolated and sequenced from DBM. The full-length of Px-hsp70cDNA was2128bp, including an1899bp open reading frame (ORF) that encoded632amino acids. The deduced protein had a computed molecular mass of69.3kDa. The Px-hsp70belonged to hsp70gene family based on its high identity of amino acid sequences to the hsp70genes from other insect species.
The basic expression levels of Px-hsp70in female and male larvae were lower than those in female and male adults in R and S DBM, in general. There were no significantly difference of Px-hsp70expression in female and male larvae between25℃and high temperature treatments (37and42℃). However, significantly increase of Px-hsp70expression was found in female and male adults of R and S DBM when the adults were pretreated with high temperature, and the extent of up-regulation of Px-hsp70expression in S DBM was significantly higher than in R DBM.
The results indicated that the fitness of the R and S DBM was inhibited significantly by high temperature, and the R DBM was inhibited much than S DBM.
The level of resistance to insecticide of DBM was influenced markedly by high temperature. Based on the field resistance monitoring, the level of resistance to insecticide decreased sharply in July. The level of resistance to avermectin in the progenies of DBM declined sharply when the insects were reared at33.5℃for one generation. The great decline of avermectin resistance in the progenies of R DBM,when the DBM were reared at high temperature for one generation, might be associated with its significantly lower fitness.
caspase是细胞凋亡过程中的关键酶,起到执行细胞凋亡的功能,本文通过同源克隆的策略和RACE技术,克隆得到一个1172bp长的小菜蛾caspase基因(Px-caspase-1),具有完整的开放阅读框,开放阅读框长903bp,编码具有300个氨基酸的caspase蛋白,分子量为33.6kDa。与其它昆虫caspase基因的氨基酸序列比对结果显示,Px-caspase-1)属于效应caspase。高温(32.1、36、40、42、45、47℃)和常温(25℃)预处理对caspase-9(起始caspase)和caspase-3(效应caspase)的酶活力及1px-caspase-1mRNA表达量变化影响的研究结果表明,常温下小菜蛾成虫体内存在caspase-9和caspase-3的酶活性及Px-caspase-1基因表达,高温能够显著诱导caspase-9和caspase-3酶活力增高以及Px-caspase-1mRNA表达上调,且随着温度的升高上调表达的高峰提前出现。此外,高温可导致小菜蛾成虫的死亡率显著增高。上述结果表明,常温25-C下细胞凋亡是小菜蛾的一种正常生命现象,而高温能够显著诱导小菜蛾发生细胞凋亡,使得细胞凋亡基因Px-caspase-1的上调表达高峰提前出现,加速小菜蛾的死亡。比较高温(32.1、40、42℃)处理不同时间敏感和抗性小菜蛾Px-caspase-1基因表达的差异,结果表明抗性小菜蛾Px-caspase-1基因在高温下的热激上调表达显著大于敏感小菜蛾。
在热胁迫下的细胞凋亡与热休克蛋白70家族相关,本文克隆得到小菜蛾hsp70基因(Px-hsp70)序列的全长cDNA。该基因的cDNA序列长2128bp,具有5'非翻译区90bp,3'非翻译区140bp,开放阅读框为1899bp,演绎的蛋白质序列为632aa,分子量为69.3kDa。通过在NCBI上进行的序列比对及功能位点分析,Px-hsp70)禹于hsp70家族成员。通过采用雌、雄幼虫和雌、雄成虫为材料,25、37、42-C预处理3h后,无论在敏感还是抗性种群中整体上,幼虫的Px-hsp70表达量均比成虫Px-hsp70的表达量低,且高温诱导后幼虫Px-hsp70的表达量均无显著变化。在成虫中,高温可显著诱导敏感及抗性小菜蛾Px-hsp70的上调表达,但是高温对敏感小菜蛾Px-hsp70的上调表达程度显著大于对抗性小菜蛾的影响。
这些结果表明,高温可显著抑制敏感和抗性小菜蛾的生理适合度,但是对抗性小菜蛾适合度的抑制作用更为显著。
高温可显著影响小菜蛾的抗药性水平。田间监测的结果表明,夏季7月高温期间小菜蛾抗药性水平急剧下降。小菜蛾在高温下(33.5℃)饲养一代后对阿维菌素抗性水平大幅下降,高温饲养一代后小菜蛾抗药性水平急剧下降可能与高温下抗性小菜蛾适合度显著低于敏感小菜蛾有关。
Plutella xylostella (diamondback moth, DBM) is a destructive cosmopolitan pest of cruciferous crops. As reported, the fitness of the insecticide-resistant (R) and-susceptible (S) DBM was decreased sharply under high temperature. Effects of heat stress on expression of caspase and hsp70genes and level of resistance to insecticide in the R and S DBM were studied by biochemistry, molecular biology and resistant bioassays. The results showed that resistance level of DBM was decreased significantly under heat stress, and this decrease was heritable.
Caspase is the key factor in apoptosis. The caspase gene, named Px-caspase-1, was isolated and sequenced from the DBM. The full-length of Px-caspase-1cDNA was1172bp, including an903bp open reading frame (ORF) that encoded300amino acids. The dedueed protein had a computed molecular mass of33.6kDa. The Px-caspase-1belonged to effector caspase based on its high identity of amino acid sequences to the effector caspase genes from other insect species.
The expression of Px-caspase-1and the enzyme activity of caspase-9and caspase-3under heat stress (32.1、36、40、42、45、47℃) were studied. The results showed that the basic levels of Px-caspase-1expression and caspase-9and caspase-3activities could be detected at25℃. However, the gene expression and the two enzyme activities increased significantly when the adults of DBM were pretreated with high temperature. With the increase of pretreatment temperature and time period, the Px-caspase-1mRNA expression and activities of caspase-9and caspase-3reached the peak value earlier under heat stress than those at25℃. In addition, high temperature could result in the increase of mortality of adult DBM. The results indicated that apoptosis was a normal physiological procession in DBM at normal temperature, and heat stress could significantly induce apoptosis in DBM and cause death of DBM.
The expression of Px-caspase-1between R and S adults DBM under heat stress (32.1、40、42℃) were studied. Significantly increase of Px-caspase-1expression was found in female and male adults of R and S DBM when the adults were pretreated with high temperature, and the extent of up-regulation of Px-caspase-Ⅰ expression in R DBM was significantly higher than in S DBM.
The heat shock protein70family were invoved in apoptosis under heat stress. The heat shock protein gene, named Px-hsp70, was isolated and sequenced from DBM. The full-length of Px-hsp70cDNA was2128bp, including an1899bp open reading frame (ORF) that encoded632amino acids. The deduced protein had a computed molecular mass of69.3kDa. The Px-hsp70belonged to hsp70gene family based on its high identity of amino acid sequences to the hsp70genes from other insect species.
The basic expression levels of Px-hsp70in female and male larvae were lower than those in female and male adults in R and S DBM, in general. There were no significantly difference of Px-hsp70expression in female and male larvae between25℃and high temperature treatments (37and42℃). However, significantly increase of Px-hsp70expression was found in female and male adults of R and S DBM when the adults were pretreated with high temperature, and the extent of up-regulation of Px-hsp70expression in S DBM was significantly higher than in R DBM.
The results indicated that the fitness of the R and S DBM was inhibited significantly by high temperature, and the R DBM was inhibited much than S DBM.
The level of resistance to insecticide of DBM was influenced markedly by high temperature. Based on the field resistance monitoring, the level of resistance to insecticide decreased sharply in July. The level of resistance to avermectin in the progenies of DBM declined sharply when the insects were reared at33.5℃for one generation. The great decline of avermectin resistance in the progenies of R DBM,when the DBM were reared at high temperature for one generation, might be associated with its significantly lower fitness.
引文
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