转几丁质酶和葡聚糖酶棉花对黄萎病的抗性及对非靶标生物的影响
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摘要
本试验以中国农业科学院棉花研究所转基因课题组提供的转双价几丁质酶和葡聚糖酶基因棉花品系61217-1（简称转双价基因抗病棉）为研究对象，受体中棉所24和转Bt基因抗虫棉鲁棉研28为对照，于2009-2010年，研究了转双价基因抗病棉花对靶标病害黄萎病的抗性、对非靶标病害、棉田主要害虫、天敌及土壤微生物的发生消长规律，旨在为建立转基因抗病棉花的环境安全评价标准提供依据。
主要结论如下：
1.几丁质酶基因和葡聚糖酶基因的导入极显著提高了棉花对靶标病害黄萎病的抗性，且整个生育期的抗性表达稳定。2年结果表明，转基因株系61217-1两年平均病指为13.7，达抗病水平，而受体材料中棉所24和Bt棉鲁棉研28分别为34.7和26.3，为耐病水平。且转基因抗病棉花在整个生育期对黄萎病的抗性均显著的高于对照材料。
2.几丁质酶基因和葡聚糖酶基因的导入还提高棉花对苗期和铃期病害的抗性。2年试验结果表明，61217-1的苗期死苗率均低于受体材料，且2010年差异达到显著水平；由铃期病害引起的烂铃率2009年、2010年均显著低于受体材料。
3.几丁质酶基因和葡聚糖酶基因的导入对早衰的影响不明显，但后期叶斑病的发生有加重趋势。61217-1花铃期叶斑病指显著高于受体材料，早衰指数高于受体中棉所24，但差异不显著，推断双价抗病基因的导入可能导致棉花早衰，从而引起叶斑病的严重发生。
4.双价抗病基因的导入能显著减轻二代棉铃虫和伏蚜和发生危害。推断几丁质酶基因和葡聚糖酶基因的介入可能提高植株体内几丁质酶和葡聚糖酶的活性，由此影响昆虫的生长发育。但转双价基因抗病棉花苗蚜的发生与中棉所24及鲁棉研28没有显著差异，落卵量较受体材料有增加趋势。表明双价基因的介入对同一种害虫的不同形态或同一种害虫的不同发生危害阶段影响不一致，可能与转基因成分在棉株体内的时空表达有关，相关的作用机制有待进一步研究。
5.61217-1在盲蝽象发生的某些时期数量低于受体棉花中棉所24；红蜘蛛的发生数量与受体比无显著差异，但在红蜘蛛发生危害严重的2010年，转基因抗病棉花红蜘蛛的发生危害有高于受体材料的趋势，由于红蜘蛛的发生与田间棉花受到干旱的危害显著相关，可能会导致红蜘蛛点片发生，该结论仍需要进一步验证。
6.与中棉所24相比，61217-1上瓢虫成虫数量两年趋势不一致，瓢虫幼虫和草蛉成虫数量减少，但差异均不显著；对小花蝽的发生数量无显著影响。
7.转双价基因棉花与受体中棉所24和对照抗虫棉鲁棉研28相比，对根际土壤的酸碱度及土壤可培养微生物的影响不大，并没有给土壤微生态带来不利因素。
综上所述，双价Chi+Glu基因的导入显著提高了受体材料的抗黄萎病性；苗病和铃病的发生减轻；伏蚜和二代棉铃虫幼虫的数量明显减少；对其他病害、虫害、天敌及土壤微生物的影响不明显。
Stable cotton variety61217-1into which was transferred the chitinase and glucanase bivalent geneby the Institute of Cotton Research in Chinese Academy of Agriculture Sciences, was studied. Theacceptor CCRI24and Lumian28, with a transgenic Bt gene, were used as controls. Between2009and2010, the chitinase-glucanase transgenic cotton line with resistance to Verticillium wilt disease wasanalyzed for its response to non-target disease (seedling disease, premature senescence, boll disease andleaf spot), insect damage in the field (aphid, red spider, capsid and cotton bollworm), and its mainnatural enemies (ladybird, floret bug and lacewing). The microbial flora of the soil was also studied.This provided a basis for the safety evaluation of transgenic cotton.
Main conclusions:
1. Resistance to Verticillium wilt disease was significantly enhanced in cotton strain61217-1.Compared with receptor CCRI24, the incidence and average disease index of line61217-1decreasedby53.5%and61.6%, respectively. The incidence and average disease index of line61217-1declined by39.1%and47.9%, compared with Lumian28carrying the Bt gene. Thus, it provided good resistance towilt disease in the field. The chitinase-glucanase transgenic cotton line was much more resistant toVerticillium wilt than the receptor material during the whole growth period, implying that thechitinase-glucanase gene was expressed stably during the whole growth period.
2. The rate of seedling death of the transgenic disease-resistant cotton was3.7–6.8percentage points lower than the receptor material, and0.6–8.2percentage points lower thanLumian28. In addition, it had a significantly lower rotten bell rate:9.8–16.6percentagepoints lower than the receptor material. Thus, the transgenic cotton not only had improved resistance toVerticillium wilt, but also showed improved resistance to seedling and boll disease.
3. At the blossoming and boll forming stage, the leaf spot disease index of cotton61217-1wassignificantly higher than receptor CCRI24and Lumian28. Moreover, the index of Premature Senilitywas slightly higher than receptor CCRI24and Lumian28. Thus, transformation of cotton with thedisease resistant bivalent gene might lead to early aging and increased susceptibility to leaf spot disease.
4. During the two years of this study, the number of summer ecotype and the second generationbollworm observed on the bivalent transgenic disease-resisting cotton was significantly lower than onCCRI24. This indicated that the presence of the bivalent transgene could significantly reduce thedamage caused by summer ecotype and second generation bollworm. There was no significantdifference between CCRI24and Lumian28in the occurrence of seedling aphids. The occurrence ofthree generations of larvae was not consistent. The number of H. armigera eggs on61217-1increasedcompared with CRI24and Lumian28. The introduction of the bivalent transgene had different effectson the various morphological stages of the same pest and the mechanism of action requires furtherstudy.
5. There were fewer capsids in the chitinase-glucanase transgenic cotton line and Lumian28thanin receptor CCRI24. The number of red spiders was not significantly different among the three strains.The damage caused by red spiders was much more severe in2010and the transgenic cotton had a tendency towards more damage compared with CCRI24. The occurrence of red spider had a significantcorrelation with cotton that was damaged by drought. Red spider might have occurred as a spot film, butthis conclusion requires further verification.
6. Compared with receptor CCRI24, the occurrence of adult ladybirds on the chitinase-glucanasetransgenic cotton line was not consistent over the two-year study period. The number of larval ladybirdsand lacewings was reduced, but not significantly. There was no effect on the presence of the Floret bugon the chitinase-glucanase transgenic cotton.
7. Compared with receptor CRRI24and Lumian28, the cotton carrying the bivalent gene had aslight, but not unfavorable, effect on soil microorganisms. During the different growth stages of cotton,the rhizosphere soil pH was nearly constant. Furthermore, the soil pH of cotton61217-1was lower thanthat of receptor CCRI24, i.e., the rhizosphere soil pH of the transgenic cotton tended towards acidity.
As mentioned above,recipient variety resistant to Verticilliumwillt was promoted significantly withreducing seeding disease and boll rot because of double gene(Chi+Glu) mediation. Besides, the numberof summer ecotype and the second generation of cotton bollworm larvae decreased obviously. While theeffects on other diseases, pest,natural enemies and soil microorganism were not obvious.
主要结论如下：
1.几丁质酶基因和葡聚糖酶基因的导入极显著提高了棉花对靶标病害黄萎病的抗性，且整个生育期的抗性表达稳定。2年结果表明，转基因株系61217-1两年平均病指为13.7，达抗病水平，而受体材料中棉所24和Bt棉鲁棉研28分别为34.7和26.3，为耐病水平。且转基因抗病棉花在整个生育期对黄萎病的抗性均显著的高于对照材料。
2.几丁质酶基因和葡聚糖酶基因的导入还提高棉花对苗期和铃期病害的抗性。2年试验结果表明，61217-1的苗期死苗率均低于受体材料，且2010年差异达到显著水平；由铃期病害引起的烂铃率2009年、2010年均显著低于受体材料。
3.几丁质酶基因和葡聚糖酶基因的导入对早衰的影响不明显，但后期叶斑病的发生有加重趋势。61217-1花铃期叶斑病指显著高于受体材料，早衰指数高于受体中棉所24，但差异不显著，推断双价抗病基因的导入可能导致棉花早衰，从而引起叶斑病的严重发生。
4.双价抗病基因的导入能显著减轻二代棉铃虫和伏蚜和发生危害。推断几丁质酶基因和葡聚糖酶基因的介入可能提高植株体内几丁质酶和葡聚糖酶的活性，由此影响昆虫的生长发育。但转双价基因抗病棉花苗蚜的发生与中棉所24及鲁棉研28没有显著差异，落卵量较受体材料有增加趋势。表明双价基因的介入对同一种害虫的不同形态或同一种害虫的不同发生危害阶段影响不一致，可能与转基因成分在棉株体内的时空表达有关，相关的作用机制有待进一步研究。
5.61217-1在盲蝽象发生的某些时期数量低于受体棉花中棉所24；红蜘蛛的发生数量与受体比无显著差异，但在红蜘蛛发生危害严重的2010年，转基因抗病棉花红蜘蛛的发生危害有高于受体材料的趋势，由于红蜘蛛的发生与田间棉花受到干旱的危害显著相关，可能会导致红蜘蛛点片发生，该结论仍需要进一步验证。
6.与中棉所24相比，61217-1上瓢虫成虫数量两年趋势不一致，瓢虫幼虫和草蛉成虫数量减少，但差异均不显著；对小花蝽的发生数量无显著影响。
7.转双价基因棉花与受体中棉所24和对照抗虫棉鲁棉研28相比，对根际土壤的酸碱度及土壤可培养微生物的影响不大，并没有给土壤微生态带来不利因素。
综上所述，双价Chi+Glu基因的导入显著提高了受体材料的抗黄萎病性；苗病和铃病的发生减轻；伏蚜和二代棉铃虫幼虫的数量明显减少；对其他病害、虫害、天敌及土壤微生物的影响不明显。
Stable cotton variety61217-1into which was transferred the chitinase and glucanase bivalent geneby the Institute of Cotton Research in Chinese Academy of Agriculture Sciences, was studied. Theacceptor CCRI24and Lumian28, with a transgenic Bt gene, were used as controls. Between2009and2010, the chitinase-glucanase transgenic cotton line with resistance to Verticillium wilt disease wasanalyzed for its response to non-target disease (seedling disease, premature senescence, boll disease andleaf spot), insect damage in the field (aphid, red spider, capsid and cotton bollworm), and its mainnatural enemies (ladybird, floret bug and lacewing). The microbial flora of the soil was also studied.This provided a basis for the safety evaluation of transgenic cotton.
Main conclusions:
1. Resistance to Verticillium wilt disease was significantly enhanced in cotton strain61217-1.Compared with receptor CCRI24, the incidence and average disease index of line61217-1decreasedby53.5%and61.6%, respectively. The incidence and average disease index of line61217-1declined by39.1%and47.9%, compared with Lumian28carrying the Bt gene. Thus, it provided good resistance towilt disease in the field. The chitinase-glucanase transgenic cotton line was much more resistant toVerticillium wilt than the receptor material during the whole growth period, implying that thechitinase-glucanase gene was expressed stably during the whole growth period.
2. The rate of seedling death of the transgenic disease-resistant cotton was3.7–6.8percentage points lower than the receptor material, and0.6–8.2percentage points lower thanLumian28. In addition, it had a significantly lower rotten bell rate:9.8–16.6percentagepoints lower than the receptor material. Thus, the transgenic cotton not only had improved resistance toVerticillium wilt, but also showed improved resistance to seedling and boll disease.
3. At the blossoming and boll forming stage, the leaf spot disease index of cotton61217-1wassignificantly higher than receptor CCRI24and Lumian28. Moreover, the index of Premature Senilitywas slightly higher than receptor CCRI24and Lumian28. Thus, transformation of cotton with thedisease resistant bivalent gene might lead to early aging and increased susceptibility to leaf spot disease.
4. During the two years of this study, the number of summer ecotype and the second generationbollworm observed on the bivalent transgenic disease-resisting cotton was significantly lower than onCCRI24. This indicated that the presence of the bivalent transgene could significantly reduce thedamage caused by summer ecotype and second generation bollworm. There was no significantdifference between CCRI24and Lumian28in the occurrence of seedling aphids. The occurrence ofthree generations of larvae was not consistent. The number of H. armigera eggs on61217-1increasedcompared with CRI24and Lumian28. The introduction of the bivalent transgene had different effectson the various morphological stages of the same pest and the mechanism of action requires furtherstudy.
5. There were fewer capsids in the chitinase-glucanase transgenic cotton line and Lumian28thanin receptor CCRI24. The number of red spiders was not significantly different among the three strains.The damage caused by red spiders was much more severe in2010and the transgenic cotton had a tendency towards more damage compared with CCRI24. The occurrence of red spider had a significantcorrelation with cotton that was damaged by drought. Red spider might have occurred as a spot film, butthis conclusion requires further verification.
6. Compared with receptor CCRI24, the occurrence of adult ladybirds on the chitinase-glucanasetransgenic cotton line was not consistent over the two-year study period. The number of larval ladybirdsand lacewings was reduced, but not significantly. There was no effect on the presence of the Floret bugon the chitinase-glucanase transgenic cotton.
7. Compared with receptor CRRI24and Lumian28, the cotton carrying the bivalent gene had aslight, but not unfavorable, effect on soil microorganisms. During the different growth stages of cotton,the rhizosphere soil pH was nearly constant. Furthermore, the soil pH of cotton61217-1was lower thanthat of receptor CCRI24, i.e., the rhizosphere soil pH of the transgenic cotton tended towards acidity.
As mentioned above,recipient variety resistant to Verticilliumwillt was promoted significantly withreducing seeding disease and boll rot because of double gene(Chi+Glu) mediation. Besides, the numberof summer ecotype and the second generation of cotton bollworm larvae decreased obviously. While theeffects on other diseases, pest,natural enemies and soil microorganism were not obvious.
引文
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