人工丝素重链（artFibH）的设计及转基因家蚕品系的构建

发布时间：2018-05-07 15:01

本文选题：蚕丝纤维 + 改性　；参考：《西南大学》2017年硕士论文

【摘要】：蚕丝是一种复杂的微细纤维集合性天然蛋白,因具优良的特性被广泛使用和研究。然而力学性能上的一些缺陷,使得蚕丝的应用范围具有局限性。因此,蚕丝纤维的改性是拓展应用振兴产业的当务之急。蚕丝纤维的改性研究已有较长历史,改性的方法可分为物理改性、化学改性和遗传改性。物理和化学的改性方法只是对蚕丝进行修饰,不能从本质上改变蚕丝蛋白特性,且成本高、工序繁琐。利用遗传操作技术对蚕丝基因进行人工改造,能够获得性能更优良且可稳定遗传的新品系。蜘蛛丝是已知动物纤维中力学性能最优越的丝纤维,其蛋白特性、组分及成丝过程与蚕丝具有相似性,因此不断有学者尝试在家蚕丝腺中表达蜘蛛丝来提高蚕丝的力学性能,但外源基因表达量低一直是无法攻克的难题。如果能通过编辑蚕丝自身的基因来实现蚕丝性能的改良则是最经济有效的途径。研究发现蜘蛛丝重复区域序列较比蚕丝的更加规则有序,这可能是蜘蛛丝强于蚕丝的重要原因。丝素重链Fib-H基因是丝蛋白最重要的结构基因,具有长片段的重复区域,决定着蚕丝力学性能。有研究者发现对Fib-H进行截短后蚕丝的力学性能变差。那么,如果人为改造丝素重链使其序列更规则,长度更长就有可能获得保持天然特性的超强人工蚕丝。丝素重链高度重复和高GC含量的特性,使得利用常规的PCR技术和测序技术难以对其进行简单地克隆及编辑。因此,我们采用人工组装基因的方法对此设想做了初步的探索。本论文取得的主要成果如下:1、人工丝素重链(artFibH)的设计根据Fib-H基因的序列特征,我们选择重复序列最长的第七个重复区(R07)和第六个非重复区(A06)作为一个重复单元,称为“A06R07”。为保证人工丝素重链保持原来的序列特征,我们选择了两个IIs型限制内切酶:Bbs I和BsaI,在基因的两条链上各含有一个Bbs I和一个BsaI的识别序列,并且均设计在质粒载体上。Bbs I识别序列设计在A06R07序列的下游,其中一个设计在“A06R07”两个碱基之后,酶切后A06R07序列露出3?端TTGT末端;Bsa I识别序列设计在A06R07序列的两端,其中一个设计在A06R07序列的上游五个碱基之前,酶切后A06R07序列形成5?端AACA末端,另一个BbsI和Bsa I识别序列之间相距7个碱基,二者共用一个酶切位点,切出的一对粘性末端正好碱基互补,利用酶切连接的方法实现重复单元之间无缝连接加倍。2、artFibH转基因家蚕品系的建立经酶切验证,我们共成功构建了由Fib-H启动子驱动人工Fib-H基因的piggyBac转基因载体如下:pBac[R01A06R07R12]、pBac[R01(A06R07)3R12]、pBac[R01(A06R07)5R12]、pBac[R01(A06R07)5-last-R12]、pBac[R01(A06R07)3-Red-R12]、pBac[R01(A06R07)3-SELR13-R12]。以家蚕实用品系932为受体材料,利用显微注射技术,制备了转基因家蚕。通过荧光观察筛选到阳性个体。其中pBac[R01(A06R07)3R12]有2个蛾圈,阳性蛾圈率为0.2%,pBac[R01(A06R07)5R12]有1个蛾圈,阳性蛾圈率为0.5%。3、artFibH转基因蚕丝的相关检测通过蛋白预测软件分析3?型和5?型的人工Fib-H蛋白大小分别约为203KDa和295KDa,均小于家蚕932品系Fib-H蛋白(350KDa)。通过SDS-PAGE银染和Western Blot检测,结果显示预测目的条带并不明显,因此推测可能是由于其蛋白分子量太大而且具有自组装的特性,而滞留在点样孔中;通过冷冻切片技术获得单根茧丝的横截面直径,结果显示野生型与人工型茧丝形态和直径并无明显差异,表明artFibH的插入不会影响茧丝的正常形态和直径大小;通过应力应变拉伸试验,结果显示,与WT型相比,人工3?型和5?型茧丝伸长率稍有提高,强度、韧性、弹性模量稍有降低,表明由于转基因插入位点的不确定性,artFibH插入后降低了蚕丝的力学性能;通过傅立叶红外光谱试验,进一步分析了茧丝蛋白二级结构,结果显示,与WT型相比,人工3?型和5?型的茧丝蛋白构象中,β-折叠、α-螺旋和无规则卷曲含量稍有提高,β-转角含量稍有降低,表明成丝过程中artFibH会促进β-折叠的形成。
[Abstract]:Silk is a kind of complex natural protein of microfiber, which has been widely used and studied because of its excellent characteristics. However, some defects in mechanical properties make the application range of silkworm silk limited. Therefore, the modification of silk fiber is an urgent task to expand the application and revitalization of the industry. The modification of silk fiber has a long history. The modification methods can be divided into physical modification, chemical modification and genetic modification. Physical and chemical modification methods only modify silkworm silk, which can not change silk protein properties in essence, and the cost is high and the process is tedious. The spider silk is the most superior silk fiber in the known animal fibers. Its protein characteristics, components and filaments are similar to silk. Therefore, some scholars have tried to express spider silk in the silk gland to improve the mechanical properties of silk, but the low expression of foreign groups has always been a difficult problem to be overcome. It is the most economical and effective way to improve silk properties by editing the genes of silk itself. It is found that the repeated sequence of the spider silk is more regular than silk, which may be the important reason why the spider silk is stronger than the silk. The Silk Fibroin Heavy Chain Fib-H gene is the most important structural gene of the silk egg white, with a long fragment. Repeat areas determine the mechanical properties of silk. Some researchers have found that the mechanical properties of silkworm silk after the truncation of Fib-H become worse. Then, if human silk fibroin heavy chain is reformed to make its sequence more regular and longer, it is possible to obtain super strong silk with natural characteristics. The high repetition of silk fibroin heavy chain and high GC content can make use of the silk fibroin heavy chain. The conventional PCR technology and sequencing technology are difficult to simply clone and edit them. Therefore, we have made a preliminary exploration of this idea by using artificial assembly genes. The main achievements of this paper are as follows: 1, the design of artificial Fibroin Heavy Chain (artFibH) is based on the sequence characteristics of the Fib-H gene, we choose the longest repeat sequence. Seventh repeat areas (R07) and sixth non repetition areas (A06) are called "A06R07". In order to keep the original sequence characteristics of the Silk Fibroin Heavy Chain, we choose two IIs type restriction endonucleases: Bbs I and BsaI, each containing a Bbs I and a BsaI identification sequence on the two chain of the gene, and all are designed in the sequence. The.Bbs I identification sequence on the plasmid vector is designed downstream of the A06R07 sequence, one of which is designed after "A06R07" two bases. After the enzyme, the A06R07 sequence reveals 3? Terminal TTGT ends; the Bsa I recognition sequence is designed at both ends of the A06R07 sequence, one of which is before the upstream five base of the A06R07 sequence, and the A06R07 sequence after the enzyme is formed to form 5? The terminal AACA terminal, another BbsI and Bsa I identification sequence between 7 bases, the two shared an enzyme cut site, a pair of sticky ends just base complementary, using the method of enzyme cut connection to realize the duplication of the seamless connection between the.2, the establishment of artFibH transgenic silkworm strains verified by enzyme cutting, we successfully constructed the The Fib-H promoter drives the piggyBac transgenic vector of the artificial Fib-H gene as follows: pBac[R01A06R07R12], pBac[R01 (A06R07) 3R12], pBac[R01 (A06R07) 5R12], pBac[R01 (A06R07). PBac[R01 (A06R07) 3R12] has 2 moth circles, the positive moth rate is 0.2%, the pBac[R01 (A06R07) 5R12] has 1 moth circles, the positive moth rate is 0.5%.3, and the correlation detection of artFibH transgenic silk is divided into 3 and 5? Type artificial Fib-H protein, respectively, is about 203KDa, respectively, and the size of the artificial Fib-H protein is about 203KDa. And 295KDa, all less than the Fib-H protein (350KDa) of the 932 line of silkworm. Through SDS-PAGE silver staining and Western Blot detection, the results showed that the predicted target strip was not obvious. Therefore, it is presumed that it may be due to the large protein molecular weight and self assembly characteristics, and it is stuck in the point like hole. The cross section of single cocoon silk is obtained by freezing section technique. The surface diameter showed that there was no significant difference between the shape and diameter of the wild type and artificial cocoon silk, indicating that the insertion of artFibH did not affect the normal form and diameter of the cocoon silk. By the stress strain tensile test, the results showed that the elongation of artificial 3? And 5? Type cocoons was slightly improved, strength, toughness, and modulus of elasticity were slightly lower than that of WT. It is shown that the artFibH insertion reduces the mechanical properties of silkworm silk because of the uncertainty of the insertion site of the transgenic plant. The two grade structure of cocoon silk protein is further analyzed by Fu Liye infrared spectroscopy. The results show that the content of beta folding, alpha helix and irregular curl in artificial 3? Type and 5? Type cocoon protein structures are slightly higher than that of WT type. The content of beta - rotation decreased slightly, indicating that artFibH promoted the formation of beta - fold during filaments.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;S881

【参考文献】