本文選題：蠶絲纖維 + 改性��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：蠶絲是一種復(fù)雜的微細(xì)纖維集合性天然蛋白,因具優(yōu)良的特性被廣泛使用和研究。然而力學(xué)性能上的一些缺陷,使得蠶絲的應(yīng)用范圍具有局限性。因此,蠶絲纖維的改性是拓展應(yīng)用振興產(chǎn)業(yè)的當(dāng)務(wù)之急。蠶絲纖維的改性研究已有較長歷史,改性的方法可分為物理改性、化學(xué)改性和遺傳改性。物理和化學(xué)的改性方法只是對蠶絲進(jìn)行修飾,不能從本質(zhì)上改變蠶絲蛋白特性,且成本高、工序繁瑣。利用遺傳操作技術(shù)對蠶絲基因進(jìn)行人工改造,能夠獲得性能更優(yōu)良且可穩(wěn)定遺傳的新品系。蜘蛛絲是已知動物纖維中力學(xué)性能最優(yōu)越的絲纖維,其蛋白特性、組分及成絲過程與蠶絲具有相似性,因此不斷有學(xué)者嘗試在家蠶絲腺中表達(dá)蜘蛛絲來提高蠶絲的力學(xué)性能,但外源基因表達(dá)量低一直是無法攻克的難題。如果能通過編輯蠶絲自身的基因來實現(xiàn)蠶絲性能的改良則是最經(jīng)濟(jì)有效的途徑。研究發(fā)現(xiàn)蜘蛛絲重復(fù)區(qū)域序列較比蠶絲的更加規(guī)則有序,這可能是蜘蛛絲強(qiáng)于蠶絲的重要原因。絲素重鏈Fib-H基因是絲蛋白最重要的結(jié)構(gòu)基因,具有長片段的重復(fù)區(qū)域,決定著蠶絲力學(xué)性能。有研究者發(fā)現(xiàn)對Fib-H進(jìn)行截短后蠶絲的力學(xué)性能變差。那么,如果人為改造絲素重鏈?zhǔn)蛊湫蛄懈?guī)則,長度更長就有可能獲得保持天然特性的超強(qiáng)人工蠶絲。絲素重鏈高度重復(fù)和高GC含量的特性,使得利用常規(guī)的PCR技術(shù)和測序技術(shù)難以對其進(jìn)行簡單地克隆及編輯。因此,我們采用人工組裝基因的方法對此設(shè)想做了初步的探索。本論文取得的主要成果如下:1、人工絲素重鏈(artFibH)的設(shè)計根據(jù)Fib-H基因的序列特征,我們選擇重復(fù)序列最長的第七個重復(fù)區(qū)(R07)和第六個非重復(fù)區(qū)(A06)作為一個重復(fù)單元,稱為“A06R07”。為保證人工絲素重鏈保持原來的序列特征,我們選擇了兩個IIs型限制內(nèi)切酶:Bbs I和BsaI,在基因的兩條鏈上各含有一個Bbs I和一個BsaI的識別序列,并且均設(shè)計在質(zhì)粒載體上。Bbs I識別序列設(shè)計在A06R07序列的下游,其中一個設(shè)計在“A06R07”兩個堿基之后,酶切后A06R07序列露出3?端TTGT末端;Bsa I識別序列設(shè)計在A06R07序列的兩端,其中一個設(shè)計在A06R07序列的上游五個堿基之前,酶切后A06R07序列形成5?端AACA末端,另一個BbsI和Bsa I識別序列之間相距7個堿基,二者共用一個酶切位點,切出的一對粘性末端正好堿基互補(bǔ),利用酶切連接的方法實現(xiàn)重復(fù)單元之間無縫連接加倍。2、artFibH轉(zhuǎn)基因家蠶品系的建立經(jīng)酶切驗證,我們共成功構(gòu)建了由Fib-H啟動子驅(qū)動人工Fib-H基因的piggyBac轉(zhuǎn)基因載體如下: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]。以家蠶實用品系932為受體材料,利用顯微注射技術(shù),制備了轉(zhuǎn)基因家蠶。通過熒光觀察篩選到陽性個體。其中pBac[R01(A06R07)3R12]有2個蛾圈,陽性蛾圈率為0.2%,pBac[R01(A06R07)5R12]有1個蛾圈,陽性蛾圈率為0.5%。3、artFibH轉(zhuǎn)基因蠶絲的相關(guān)檢測通過蛋白預(yù)測軟件分析3?型和5?型的人工Fib-H蛋白大小分別約為203KDa和295KDa,均小于家蠶932品系Fib-H蛋白(350KDa)。通過SDS-PAGE銀染和Western Blot檢測,結(jié)果顯示預(yù)測目的條帶并不明顯,因此推測可能是由于其蛋白分子量太大而且具有自組裝的特性,而滯留在點樣孔中;通過冷凍切片技術(shù)獲得單根繭絲的橫截面直徑,結(jié)果顯示野生型與人工型繭絲形態(tài)和直徑并無明顯差異,表明artFibH的插入不會影響繭絲的正常形態(tài)和直徑大小;通過應(yīng)力應(yīng)變拉伸試驗,結(jié)果顯示,與WT型相比,人工3?型和5?型繭絲伸長率稍有提高,強(qiáng)度、韌性、彈性模量稍有降低,表明由于轉(zhuǎn)基因插入位點的不確定性,artFibH插入后降低了蠶絲的力學(xué)性能;通過傅立葉紅外光譜試驗,進(jìn)一步分析了繭絲蛋白二級結(jié)構(gòu),結(jié)果顯示,與WT型相比,人工3?型和5?型的繭絲蛋白構(gòu)象中,β-折疊、α-螺旋和無規(guī)則卷曲含量稍有提高,β-轉(zhuǎn)角含量稍有降低,表明成絲過程中artFibH會促進(jìn)β-折疊的形成。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;S881

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