家蚕翅原基发育的组织形态学观察及BmSRF基因的原位杂交
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摘要
家蚕是完全变态昆虫,其成虫的一些器官是由成虫盘发育而来。翅原基是家蚕成虫盘之一,位于第2和第3胸节的两侧,分别发育为成虫的前后翅。在家蚕的一生,尤其在幼虫到蛹以及蛹到成虫的变态过程中,翅原基发生着很大的变化。家蚕有着十分丰富的遗传突变资源。在家蚕翅发育形态学方面的研究已有报道,但对于其他突变体的形态变化所知不多,他们在组织形态学上到底差异如何我们还不清楚。
本论文通过对家蚕正常型(大造)与突变型(cf)材料进行解剖测量,初步获得了上簇前翅原基在宏观上的变化规律,找出了两种材料翅原基在宏观上发育的不同;利用石蜡切片技术,获得家蚕翅原基不同发育时期的显微实体照片,印证补充了前人关于家蚕翅原基发育的形态学研究,为家蚕翅原基发育基因功能研究提供形态学参考照片;克隆了可能与cf突变体形成有关的基因BmSRF,以家蚕大造5龄3天的翅原基为材料,以克隆的BmSRF基因标记cDNA探针,获得了家蚕BmSRF基因组织定位信息。研究结果如下:
1.以家蚕正常型(大造)与突变型(cf)翅原基为材料对其进行解剖,利用imageJ测量软件进行测量,获得其发育规律,找出了两种材料翅原基在宏观上发育的不同以及差异时期。大造翅原基在五龄2d到3d与4d到5d及8d到上簇时期变化最大,而对于cf突变体翅原基在五龄2d到3d与4d到5d及6d到上簇时期变化最大。
2.获得了家蚕五龄各时期翅原基发育的实体照片,观察到了翅原基开始伸展的时期并获得了其向外伸展过程的连续图片,区分了与翅原基发育相关的各个组织,如肌肉组织、气管、结缔组织等组织图片。比较了正常型、突变型翅原基发育的不同,发现此突变型(cf)较正常型没有本质上的差异,都有气管丛的出现、翅芽的伸展等。不同的是在翅芽的大小上正常型的要小于突变型的;另一差异在于,正常型翅囊5龄3d时与翅芽分开且变薄,5龄4d翅芽向外展开,而突变型的翅芽5龄2d开始展开,5龄4d时翅囊变薄。
3.以家蚕5龄3d翅原基cDNA为模板,克隆了翅原基发育相关基因BmSRF,对此基因进行了生物信息学分析,发现家蚕中的SRF同源基因由3个外显子组成,ORF为906bp,编码了302个氨基酸。该基因在整个翅发育时期都有表达。在幼虫期,从5龄1天起至预蛹期,其表达量逐渐降低,后表达量慢慢升高,到化蛹第1天(P1),其表达量骤然提高。
4.利用已初步建立的原位杂交体系对家蚕BmSRF基因进行了定位,结果表明家蚕BmSRF基因仅在翅原基组织中表达,而对于其发育相关组织如气管、肌肉等没有信号。
The silkworm (Bombyx mori) is the holometabolous insect and some of its adult organs come from imaginal discs. The wing disc is one of the imaginal discs of the silkworm, which locates the two sides of the 2nd and 3rd thoracic section and grows separately into adult wing. The wing disc changes greatly in the whole life of the silkworm, especially during the metamorphism of the larva to the pupae, as well as the pupae to adult. There are abundant mutant resources of in the silkworm. Although the morphology of the wing disc has been reported, it is very short in the developmental process and differentiation between wild type and mutants.
Through dissecting and measuring the wild type (dazao) and mutant (cf) of wing disc, we obtained the macroscopical character of the wing disc development in the pre-wandering stage and identified the differences between two materials. Using paraffin sections technology, we obtained the microscopic entity photos of the silkworm wing disc in different stages and confirmed and supplied the studies of the silkworm wing disc. These results could be provided morphology reference for wing disc developmental gene function research in the silkworm. Then, we cloned the BmSRF gene, which may be related to cf mutant of the silkworm and in situ hybridized with BmSRF gene on day 3 of the 5th instar larvae.
The main research contents and results are as follows:
1. We dissected the wing disc of the wild type (dazao) and mutant (cf) and measured its length using imageJ software. The morphological and development-stage differences of wing disc were found, and the macroscopical character was obtained. The greatest changes of wing disc morphology in Dazao strain were found in the 2nd～3rd, 4th～5th day of the fifth instar and 8th day of the fifth instar larvae to wandering stage. While, in cf mutant, the stages of it's greatest changes are the 2nd～3rd, 4th～5th day of the fifth instar and 6th day of the fifth larvae to wandering stage.
2. Through the paraffin sections for the wing disc of the fifth instar larvae, its entity photos in each day of the fifth instar larvae were obtained. The beginning time of wing disc extension was observed, and the sequential photos of extension course were obtained. And we differentiated between the wing disc and related organs, such as muscle, trachea, and connective tissue. We found that the wing disc had not natural differences between wild type and cf mutant, which both had many tracheas and extension of wing bud, etc. However, the wing bud of cf mutant was larger than that of wild type. On the other hand, for the normal wing, the wing sac became thinner and dissected with the wing bud on day three of the fifth instar, and the wing bud extended on day four of the fifth instar. But, for the cf mutant, the wing bud extended on day two of the fifth instar, and the wing sac became thinner on day fourth.
3. The silkworm BmSRF gene was cloned from the wing disc on day three of the fifth instar larvae. Sequence analysis indicated that the length of coding sequence of BmSRF is 906 bp and codes a protein of 302 residues, which contained 3 exons. In our experiment, the expression signal of BmSRF was detected during the fifth instar larvae to pupae. The expression level was gradually decreased from the 1st day of the fifth instar larvae to wandering stage, but sharply increased in one-day pupae.
4. Using the in situ hybridization, the silkworm BmSRF gene was located. The result indicated that the BmSRF expressed only in the wing disc. While the expression signal of BmSRF was not detected in trachea, the muscle.
本论文通过对家蚕正常型(大造)与突变型(cf)材料进行解剖测量,初步获得了上簇前翅原基在宏观上的变化规律,找出了两种材料翅原基在宏观上发育的不同;利用石蜡切片技术,获得家蚕翅原基不同发育时期的显微实体照片,印证补充了前人关于家蚕翅原基发育的形态学研究,为家蚕翅原基发育基因功能研究提供形态学参考照片;克隆了可能与cf突变体形成有关的基因BmSRF,以家蚕大造5龄3天的翅原基为材料,以克隆的BmSRF基因标记cDNA探针,获得了家蚕BmSRF基因组织定位信息。研究结果如下:
1.以家蚕正常型(大造)与突变型(cf)翅原基为材料对其进行解剖,利用imageJ测量软件进行测量,获得其发育规律,找出了两种材料翅原基在宏观上发育的不同以及差异时期。大造翅原基在五龄2d到3d与4d到5d及8d到上簇时期变化最大,而对于cf突变体翅原基在五龄2d到3d与4d到5d及6d到上簇时期变化最大。
2.获得了家蚕五龄各时期翅原基发育的实体照片,观察到了翅原基开始伸展的时期并获得了其向外伸展过程的连续图片,区分了与翅原基发育相关的各个组织,如肌肉组织、气管、结缔组织等组织图片。比较了正常型、突变型翅原基发育的不同,发现此突变型(cf)较正常型没有本质上的差异,都有气管丛的出现、翅芽的伸展等。不同的是在翅芽的大小上正常型的要小于突变型的;另一差异在于,正常型翅囊5龄3d时与翅芽分开且变薄,5龄4d翅芽向外展开,而突变型的翅芽5龄2d开始展开,5龄4d时翅囊变薄。
3.以家蚕5龄3d翅原基cDNA为模板,克隆了翅原基发育相关基因BmSRF,对此基因进行了生物信息学分析,发现家蚕中的SRF同源基因由3个外显子组成,ORF为906bp,编码了302个氨基酸。该基因在整个翅发育时期都有表达。在幼虫期,从5龄1天起至预蛹期,其表达量逐渐降低,后表达量慢慢升高,到化蛹第1天(P1),其表达量骤然提高。
4.利用已初步建立的原位杂交体系对家蚕BmSRF基因进行了定位,结果表明家蚕BmSRF基因仅在翅原基组织中表达,而对于其发育相关组织如气管、肌肉等没有信号。
The silkworm (Bombyx mori) is the holometabolous insect and some of its adult organs come from imaginal discs. The wing disc is one of the imaginal discs of the silkworm, which locates the two sides of the 2nd and 3rd thoracic section and grows separately into adult wing. The wing disc changes greatly in the whole life of the silkworm, especially during the metamorphism of the larva to the pupae, as well as the pupae to adult. There are abundant mutant resources of in the silkworm. Although the morphology of the wing disc has been reported, it is very short in the developmental process and differentiation between wild type and mutants.
Through dissecting and measuring the wild type (dazao) and mutant (cf) of wing disc, we obtained the macroscopical character of the wing disc development in the pre-wandering stage and identified the differences between two materials. Using paraffin sections technology, we obtained the microscopic entity photos of the silkworm wing disc in different stages and confirmed and supplied the studies of the silkworm wing disc. These results could be provided morphology reference for wing disc developmental gene function research in the silkworm. Then, we cloned the BmSRF gene, which may be related to cf mutant of the silkworm and in situ hybridized with BmSRF gene on day 3 of the 5th instar larvae.
The main research contents and results are as follows:
1. We dissected the wing disc of the wild type (dazao) and mutant (cf) and measured its length using imageJ software. The morphological and development-stage differences of wing disc were found, and the macroscopical character was obtained. The greatest changes of wing disc morphology in Dazao strain were found in the 2nd～3rd, 4th～5th day of the fifth instar and 8th day of the fifth instar larvae to wandering stage. While, in cf mutant, the stages of it's greatest changes are the 2nd～3rd, 4th～5th day of the fifth instar and 6th day of the fifth larvae to wandering stage.
2. Through the paraffin sections for the wing disc of the fifth instar larvae, its entity photos in each day of the fifth instar larvae were obtained. The beginning time of wing disc extension was observed, and the sequential photos of extension course were obtained. And we differentiated between the wing disc and related organs, such as muscle, trachea, and connective tissue. We found that the wing disc had not natural differences between wild type and cf mutant, which both had many tracheas and extension of wing bud, etc. However, the wing bud of cf mutant was larger than that of wild type. On the other hand, for the normal wing, the wing sac became thinner and dissected with the wing bud on day three of the fifth instar, and the wing bud extended on day four of the fifth instar. But, for the cf mutant, the wing bud extended on day two of the fifth instar, and the wing sac became thinner on day fourth.
3. The silkworm BmSRF gene was cloned from the wing disc on day three of the fifth instar larvae. Sequence analysis indicated that the length of coding sequence of BmSRF is 906 bp and codes a protein of 302 residues, which contained 3 exons. In our experiment, the expression signal of BmSRF was detected during the fifth instar larvae to pupae. The expression level was gradually decreased from the 1st day of the fifth instar larvae to wandering stage, but sharply increased in one-day pupae.
4. Using the in situ hybridization, the silkworm BmSRF gene was located. The result indicated that the BmSRF expressed only in the wing disc. While the expression signal of BmSRF was not detected in trachea, the muscle.
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