【作者】 于振诚；

【导师】 牟志美；

【作者基本信息】 山东农业大学 ， 森林培育， 2008， 博士

【摘要】 家蚕茧的荧光色与性别有关,采用荧光选茧,定向培育等育种手段,已成功育成依据蚕茧荧光色可判别性别的荧光茧色性连锁中国系统蚕品种“荧限8417”、“荧光”和日本系统蚕品种“春玉”。组配的一代杂交组合“荧光×春玉”通过国家特殊蚕品种审定,这在国内外尚属首次。但,其性连锁遗传机理及雌雄间荧光色素代谢差异等,目前均尚不清楚。解明其遗传机理及雌雄荧光色素代谢差异,对丰富家蚕遗传学及荧光色素代谢理论、指导家蚕荧光茧色性连锁育种实践等,均有重大的学术和实用价值。本研究对蚕茧荧光色的特征特性,重点对荧光茧色性连锁蚕品种荧光色素的来源、雌雄代谢差异、遗传机理及分子标记等进行了系统的试验研究,主要结果如下。1.资源蚕品种蚕茧的荧光色可分为“黄白荧光茧色型”、“兰紫荧光茧色型”和“混合荧光茧色型”三大荧光茧色类型;同一品种的蚕茧荧光色,有明显的雄性偏黄白,雌性偏兰紫的趋向性;同品种高温干燥条件下上蔟,多营黄白荧光色茧,而在高温多湿条件下上蔟,则多营兰紫荧光色茧;黄白荧光色茧的丝胶的水溶性快于兰紫荧光色茧的水溶性;选雌性荧光兰色茧个体与雄性荧光黄色茧个体杂交,经历代选育,可育成荧光茧色性连锁蚕品种。2.从5龄起蚕或3日后开始用70%乙醇浸提过的桑叶粉和未经处理的桑叶粉配制的人工饲料饲育荧光茧色性连锁蚕品种,结果:饲以未经处理的桑叶粉配制的人工饲料所营的蚕茧紫外线下可判别雌雄;而饲以70%乙醇浸提过的桑叶粉配制的人工饲料所营蚕茧雌雄均为均匀的紫荧光色,利用蚕茧的荧光色已无法分辨雌雄。这一方面说明雌雄蚕茧荧光色差异的荧光色素基础物质来源于桑叶醇溶性物质;另一方面证明了雌雄荧光色的差别产生在5龄3日后;同时,也证明了荧光黄色素决定该类品种雌雄蚕茧荧光色的差异,为此将其作为荧光主色素研究。3.纸上色层分析和HPLC分析结果都证明,构成蚕茧荧光色的色素种类,雌雄间并不存在显著的差异。造成雌雄荧光茧色不同的原因主要是荧光黄色素的含量不同,是荧光色素量的差别,并非质的差别;雌雄中部丝腺相互移植的结果证实了中部丝腺的荧光色素直接来源于血液,中部丝腺对荧光色素无选择吸收、透过功能。4.蚕茧中除具有中部丝腺由来的荧光色素外,还有一种Rf值为0.77的紫荧光色素,血液和中部丝腺均未检出,说明蚕在吐丝营茧过程中荧光物质有新的变化,这也许是家蚕荧光茧色易受蔟中条件影响而发生变化的主要原因之一。5.在荧光茧色可判断性别中起决定性作用的荧光黄色素,雌雄出现显著差异是在5龄4日后。表现为: 4日后,随日龄的增加,与血液、中部丝腺、蚕茧雌性呈紫荧光色,雄性呈黄荧光色相反,雌性中肠的荧光黄色不断加深,5日后呈鲜艳的明黄色直至熟蚕,而雄性整个5龄期中肠的主要部分都呈褐色。说明雄性的荧光黄色素在中肠的形成、透过、释放到血液等过程都能顺利的进行,而雌性中肠虽能形成荧光黄色素,但不能顺利透过释放到血液中去,而是沉积于中肠内。可见,荧光黄色素不能顺利透过雌性中肠,导致雌性血液中荧光黄色物质总量减少,是雌雄荧光茧色产生差异的主要原因。因而,从理论上对雌茧荧光紫色、雄茧荧光黄色的荧光茧色性连锁现象和同一品种的蚕茧荧光色雌性偏兰紫雄性偏黄白现象,做出了合理的解释。6.该类荧光色素对中浓度醇类溶剂具有良好的溶解性,而对高浓度的醇溶剂溶解性较差。HPLC分析结果可见,以甲醇为流动相,他们早早被洗脱下来,吸收峰出现时间较早。说明这些成分的极性高,亲水性强。对荧光黄色素进行分离、纯化并做质谱分析,得该色素的分子量为453,分析其可能是含有一个5碳糖基的黄酮类色素。7.杂交遗传研究证明,对荧光茧色判断性别起绝对作用的荧光黄色素,为数量性状遗传,由多对基因控制,且控制该性状的基因主要存在于常染色体上;造成荧光茧色性连锁蚕品种雌雄荧光茧色不同的原因,是雄性蚕茧中的荧光黄色素显著多于雌茧。而这种差别是由于与雄性相比雌性中肠不能有效地透过该种色素,而是富集于中肠中。富集的原因有两种可能,一是缺少转运载体,二是与某种物质结合后,无法透过中肠。因雄性不存在这个问题,所以不论哪种原因都与决定雌性的W染色体有关,是由于存在于W染色体上的某种基因作用的结果。因此,虽表现差异在雄性,但根本原因在雌性,所以荧光茧色性连锁的遗传应属限性遗传类型。8.经大量分子标记引物的筛选,筛选出的ISSR-11引物的扩增条带,在大小为1541bp位点,“荧光”、“春玉”两个具有荧光茧色性连锁特性的蚕品种,雄性都扩增出一条特异性条带,而雌性在相同位点的特异性条带极浅。前面的研究已经证明了,造成荧光茧色性连锁蚕品种雌雄蚕茧荧光色不同的主要原因,是雌雄荧光黄色素的含量不同(雄性多,雌性少),ISSR-11引物雌雄扩增条带的差异,能合理解析荧光茧色性连锁的特性,可作荧光茧色性连锁类特殊蚕品种雌雄之间差异的ISSR标记。更多还原

【Abstract】 The fluorescence color of silkworm cocoon is related to the sex, we have succeeded in breeding the sex-linked cocoon fluorescence color varieties, including Chinese strain“Yingguang”、“Yingxian 8417”and Japanese strain“Chunyu”, by the fluorescence sorting of cocoon and directive breeding etc methods. F1 cross combination "Yingguang×Chunyu" has passed varieties examination and been approved by the national special silkworm, this is the first time both at home and abroad. However, the genetic mechanism of the sex-linked and fluorescent pigment metabolism differences between male and female and so on are not clear yet. Solving these questions will have important academic and practical value both in richening silkworm fluorescent pigment metabolism genetics theory and guiding the breeding practice of the sex-linked cocoon fluorescence color variety and so on.We studied the cocoon fluorescence color characteristics, and focused on the sources of fluorescent pigment in the sex-linked cocoon fluorescence color varieties\male and female differences in metabolism\genetic mechanism and molecular markers etc, the main results were as follows.1. The cocoon fluorescence color of varieties keeping in our institute could be divided into the "yellowish white cocoon fluorescence color", " bluish purple cocoon fluorescence color" and " mixed cocoon fluorescence color " three types; for the same silkworm variety, the cocoon fluorescence color of male tended to yellowish white and female tended to bluish purple clearly; silkworms of the same variety cocooning under the environment of high temperature and low humidity, the cocoons with yellowish white fluorescence color were more, and under high temperature and humidity environment, the cocoons with bluish purple were more; the water solubility of sericin of cocoon with yellowish white fluorescent color was faster than the bluish purple; the sex-linked cocoon fluorescence color variety could be bred by hybridizing female with blue fluorescence color and male with yellow, and breeding for years.2. The sex-linked cocoon fluorescence color variety was reared by the artificial diets, which were prepared by mulberry leaf powder and mulberry leaf powder extracted with 70% ethanol respectively, from the newly molted silkworm or the third day of the fifth instar. The results showed: Feeding by the artificial diet with untreated mulberry leaf powder, the female and male cocoon could be distinguished under ultraviolet light; however both the fluorescence cocoon color of female and male were even purple when feeding by the artificial diet with mulberry leaf powder extracted with 70% ethanol, and it was unable to distinguish the female and male through cocoon fluorescence color. In one hand this showed that the basis material of fluorescent pigment resulting in the cocoon fluorescence color difference between male and female stemmed from the alcohol solubility material in mulberry leaf; on the other hand the difference between male and female cocoon fluorescence color might be appeared after the third day of the fifth instar. At the same time, it proved that such differences were determined by yellow pigment, so we decided to study it as the main fluorescent pigment.3. Both the results of the paper chromatography and HPLC analysis showed that the fluorescence pigment types had no significant difference between male and female. It was the different value, especially the yellow fluorescence color, causing the difference of female and male, the difference was in quantity not in quality; results of mutual transplanting the female and male silk gland confirmed that the fluorescence pigments of middle silk gland came from the blood directly, the middle silk gland had no selective absorption and permeability to the fluorescent pigment.4. In addition to the fluorescent pigment originated from middle silk gland, there was a kind of purple fluorescent pigment with the Rf value of 0.77 in the cocoon, both of the above didn’t test from blood and middle silk gland. It showed that there was new change of fluorescent material in cocooning process, this might be one of the main reasons of the silkworm cocoon fluorescence color was easy to be influenced by the mounting conditions.5. The yellow fluorescent pigment which played decisive role in distinguishing the female and male by cocoon fluorescence color would be appeared after the fourth day of fifth instar. As follows: after the fourth day of fifth instar, the fluorescence colors of female and male in blood, middle silk gland and cocoon were purple and yellow respectively with increasing days. On the contrary, yellow fluorescence color in the female midgut deepened continuously and it was bright yellow from the fifth day of fifth instar to mature silkworm, however the main part of male midgut was brown during the entire fifth instar. It showed that the yellow fluorescence color of male in midgut could be formed, penetrated and released into blood successfully. However in female the synthetic had no problem, it couldn’t be penetrated and released into the blood smoothly, accumulated in midgut instead. It was the main reasons for the difference between male and female cocoon fluorescence color that yellow fluorescent pigment could not be penetrated through the female midgut smoothly leading to the reduce of the total amount of yellow fluorescent material in the female blood clearly. We made a reasonable explanation on the theory to the cocoon fluorescence color sex-linked phenomena of purple in female and yellow in male and to another phenomena of the male tending to yellowish white and female tending to bluish purple in the same variety.6. This type of fluorescent pigment had good solubility in the solvent of moderate concentration of alcohol, but poor in the high concentration. The results of HPLC analysis were: using methanol as the mobile phase, it could be eluded down early, and the absorption peaks appeared earlier. The polarity of these components was high and hydrophilic. We knew the molecular weight of the pigment was 453 by the separation, purification and mass spectrometry analysis of fluorescent yellow pigment, and it might contain flavonoids pigment with a five-carbon sugar.7. Study on hybrid genetic indicated that the yellow fluorescent pigment, which played decisive role in distinguishing the female and male, were quantitative genetic, and controlled by multiple genes, the genes which controlled this character were mainly on regular chromosome; The reason that caused different cocoon fluorescence color between male and female of fluorescence sex-linked varieties was that the yellow fluorescent pigment in male silkworm cocoon was more than female significantly. This difference was that this kind of pigment in the female could not penetrate through the midgut effectively, compared to the male, but accumulated in the midgut. There were two possible reasons, the first might lack of small transporters, and the second might combine with a substance, so could not penetrate through the midgut. Because that male silkworm did not have this problem, so both reasons were related to the W chromosome which determined the female, it was one kind of gene on W chromosome brought the result. Therefore, although differences showed in the male, but the basic reason was on the female, so the genetic of the sex-linked cocoon fluorescence color should belong to the genetic type of sex-limited.8. With the large number of molecular markers, the results showed that molecular marker ISSR-11 was linked to the fluorescent sex-linked and amplified bands in the molecular weight of 1541 bp sites. "Yingguang", "Chunyu," the two silkworm varieties with the sex-linked cocoon fluorescence color had amplified a specific band in the male silkworm, but females in the same site with the specificity of the very shallow. Previous studies had proved, the main reason of causing differences between female and male of the sex-linked cocoon fluorescence color varieties was the different of yellow pigment quantity in female and male (more in male, less in female). ISSR-11 primers amplified bands of the male and female differences could analysis the characteristics of fluorescence cocoon color sex-linked reasonably. It could use ISSR marker for the difference between male and female of the sex-linked cocoon fluorescence color silkworm varieties.更多还原