【正文】 在不足， 其 在轉(zhuǎn)基因植物中 表達(dá)量太低，不能滿足抗蟲 所 需要 的表達(dá)量 ，因此在實(shí)際應(yīng)用中還有一定困難。目前已經(jīng)有 多種植物導(dǎo)入 了 絲氨酸 蛋白酶抑制劑基因 ， 其中豇豆胰蛋白酶抑 制劑（ CpTI）是 抗蟲效果 最為有效的 絲氨酸 蛋白酶抑制劑，現(xiàn)已 有多種植物如 棉花 [13]、煙草 [14]、水稻 [15]等 導(dǎo)入了 CpTI 基因 ，獲得的轉(zhuǎn)基因植株 對(duì)鱗翅目和鞘翅目害蟲 均具 有較好的抗蟲效果。在植物中 ，目前 發(fā)現(xiàn)的蛋白酶抑制劑主要有三類 :絲氨酸蛋白酶抑制劑、金屬蛋白酶抑制劑 、巰基蛋白酶抑制劑 [11]。 3 表 目前發(fā)現(xiàn)的一些 Bt 毒蛋白大體分類及殺蟲譜 The general classification of Bt δendotoxin found currently and insecticidal spectrum 種類 毒蛋白名稱 殺蟲范圍 CryⅠ CryIA(a) CryIA(b) CryIA(c) CryIB CryIB(b) CryIC CryID CryIE CryIF CryIG CryIX 鱗翅目 CryⅡ CryIIA CryIIB CryIIC 鱗翅目、雙翅目 CryⅢ CryⅢ A CryⅢ B CryⅢ B2 CryⅢ C CryⅢ D 鞘翅目 CryIV CryIVA CryIVB CryIVC CryIVD 雙翅目 CryV CryVA(a) CryVA(b) CryVB CryVC 鞘翅目、鱗翅目 Cyt CytA 雙翅目 蛋白酶抑制劑基因 蛋白酶抑制劑 (Proteinase Inhibitor， PI)是 植物本身起防御作用的 并且是自然界 含量最豐富的 蛋白質(zhì)種類之一，而且某些純化的蛋白酶抑制劑對(duì)昆蟲的生長和發(fā)育具有明顯地抑制作用 [9]。在同一基因型下根據(jù)限制性內(nèi)切酶的酶譜和分子量的大小，又分為 a， b， c 等不同的基因亞型 [7]。根據(jù)它們殺蟲譜的范圍，可將 Bt 殺蟲基因分成六大類，即 CryⅠ 、 CryⅡ 、 CryⅢ 、CryⅣ 、 CryⅤ 和 Cyt。 ICPs 與昆蟲腸道細(xì)胞表面受體相互作用的特異性決定了 Bt 毒蛋白的抗蟲譜。 （ 2） Bt 毒蛋白基因分類 由于蘇云金芽孢桿菌殺蟲蛋白基因具有多樣性， Bt 毒蛋白的基因分類一直是細(xì)菌學(xué)家 們 爭論不休的問題，直到 1975 年才正式列為一個(gè)獨(dú)立的種，種以下又根據(jù)鞭毛抗原的血清型和生理生化特征的不同分為不同的亞種，后來由于無鞭毛亞種的出現(xiàn)，又把營養(yǎng)細(xì)胞的酯酶型作為輔助分類依據(jù) [6]。蘇云金芽孢桿菌殺蟲晶體蛋白（ Insecticide crystal proteins， ICPs）又稱 δ內(nèi)毒素 ， 是一類在蘇云金芽孢桿菌芽孢形成期產(chǎn)生的具有特異殺蟲活性的晶體蛋白，其在伴孢晶體內(nèi)以原毒素的形式存在 。 抗蟲基因的種類及特點(diǎn) 目前 我們 已經(jīng)發(fā)現(xiàn)并獲得了很多種抗蟲基因， 這些抗蟲基因 根據(jù) 來源進(jìn)行分類， 大體可以分為 以下 三類 ：第一類是從細(xì)菌如蘇云金芽孢桿菌中 分離得到的抗蟲基因，主要是 Bt 毒蛋白基因；第二類是從植物中分離出來的基因，包括 2 蛋白酶抑制劑基因，外源凝集素基因，淀粉酶抑制劑基因等；第三類是從動(dòng)物體內(nèi)分離出來 的 毒素基因，如 蜘蛛毒素基因和蝎毒素基因 等 [4]。通過基因工程手段 轉(zhuǎn)入外源抗蟲基因， 培育抗蟲新品種可從根本上解決問題， 而且對(duì)哺乳動(dòng)物和一些有益昆蟲不產(chǎn)生毒害作用，也 不 會(huì) 造成環(huán)境污染， 在 生產(chǎn)上 具有廣闊的應(yīng)用前景。 化學(xué)農(nóng)藥的施用在減少蟲害的同時(shí)還伴隨著一些副作用，如提高 生產(chǎn) 成本、污染 生態(tài) 環(huán)境、危害人體健康、 破壞生態(tài)系統(tǒng)、誘 導(dǎo)害蟲產(chǎn)生抗藥性、 殺死害蟲的天敵、 引起次要害蟲的大發(fā)生等，從而使 化學(xué)農(nóng)藥控制蟲害的方法在生產(chǎn)應(yīng)用受到了一定的限制。 因此，減少病蟲危害是 降低生產(chǎn)成本、 提高水稻產(chǎn)量的一個(gè)重要途徑。 同時(shí)， 水稻也是蟲害最多的糧食作物之一，田間害蟲種類達(dá) 624 種以上，每年 僅由于螟蟲危害所造成的直接經(jīng)濟(jì)損失就達(dá) 億元，使用化學(xué)殺蟲劑防治螟蟲的費(fèi)用每年達(dá) 億元，所以每年由稻螟 蟲 危害引起的總經(jīng)濟(jì)損失就能達(dá)到 億元 [3]。據(jù)統(tǒng)計(jì)，到 2025 年水稻產(chǎn)量需增加60%才能滿足日益增長的人口需求 [2]。 Pestresistant。 R818 was only transformed into the gene of were 233 transgenic plants, but only 46 plants of them were transgenic plants identified by PCR and Hyg B solution. Key words: Indica rice。s population taking it as their staple food. However， insect damage is one of the main reasons to decrease the rice yield. There are a large variety of rice pests in China. The rice production loss is more than 5% of total yield because of insect damage. At present, the main method to control rice pest is to utilize chemical pesticides. However, it caused a lot of problems, such as environmental pollution, higher production cost, food safety, etc. Breeding new rice varieties that produce insecticidal proteins by themselves is undoubtedly the most economical and security strategy. On the basis of previous reports, it is difficult to solve the problem by traditional breeding because of rice germplasm resources are scarce. Given this, it is more important to use geic engineering to breed resistant rice. In recent years, pest resistant rice with Bt genes in production have already been on a wide range of applications. With largescale cultivation of insectresistant transgenic rice, Bt gene zoophobous spectrum is relatively narrow and tolerance to pests and other problems have been attracted the attention of people. Therefore, separating and cloning new pest resistant genes from the natural world became more and more effective methods to solve these problems. Indica rice is a major subspecie of Asian cultivated rice, and more than 80% of the rice cultivars in the world belongs to indica rice, so it is important significance to study tissue culture. Tissue culture of indica rice is generally poor and regeneration rate is very low, causing some production value on a large varieties of transformation are still difficult to successfully transform or frequency low. Therefore, the study of high quality indica rice callus formation and plant regeneration ability of geic transformation rate is necessary. Agrobacterium tumefaciensmediated transformation has technique advantages including low copies, and is widely used in the study of insectresistant transgenic rice. However, the relatively large geic differences between different varieties of rice, and the transformation strong dependence on rice genotypes, increase plexity and randomness of the Agrobacterium tumefaciensmediated transformation in rice. Therefore, the optimization of Agrobacterium tumefaciensmediated rice transformation system to V improve transformation rates is necessary. This research has the following main areas of work: 1. Seqences of new Bt genes Cry30Fa1 and Cry54Aa1 that had been added cleavage site sequences at both ends, were sent to gene pany to synthesis. Then all of target gene fragments and middle cloning vector pUPROK were cut separately by two enzymes and connected to form middle carriers of the target gene fragments. Positive vectors were got by sequences identification, and were digested by endonuclease to separate target gene fragments. Then target gene fragments were inserted into pCDMARHyg vector to form eventual expression binary vectors pCDMARCry30Fa1Hyg and pCDMARCry54Aa1Hyg. All of positive plasmids were identified by digestion and sequencing. 2. By optimizing callus subculture time and different concentration of Ascorbic acid (Vc) in subculture media, callus growth state was adjusted and embryo callus were increased. Results showed that when R125 and R818 callus were subcultured 2 to 3 times, the differentiation rate was the highest。結(jié)果表明 Cry30Fa1 與Cry54Aa1 轉(zhuǎn) R125 未獲得轉(zhuǎn)基因陽性植株； Cry54Aa1 轉(zhuǎn) R818 也未獲得轉(zhuǎn)基因陽性植株； Cry30Fa1 轉(zhuǎn) R818 共獲得 233 株轉(zhuǎn)基因再生植株，經(jīng)過潮霉素溶液檢測和分子檢測，其中 46 株為含目的基因的陽性植株。結(jié)果表明洗菌后進(jìn)行干燥及分化前愈傷組織在濾紙上干燥 2 天，能夠加快分化速度，提高分化率。結(jié)果表明 R125 與 R818的愈傷組織繼代 23 次，分化率最高；繼代培養(yǎng)基中加入 Vc 濃度為 40mg/L，能減少愈傷組織褐化率且對(duì)其分化能力無影響。測序鑒定得到序列正確的陽性載體，用內(nèi)切酶切下目的基因片段并插 pCDMARHyg 載體，最終獲得高效表達(dá)雙元載體pCDMARCry30Fa1Hyg 與 p