本文關鍵詞：紅掌遺傳轉(zhuǎn)化體系與抗菌肽基因表達載體的構(gòu)建　出處：《蘇州大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： 紅掌 抗菌肽基因 再生體系 遺傳轉(zhuǎn)化 農(nóng)桿菌介導轉(zhuǎn)化法

【摘要】：紅掌(Anthurium andraeanum)為天南星科花燭屬多年生草本花卉,因其花葉形態(tài)奇特,花期持久,常作為切花材料;盆栽品種株型優(yōu)美,是廣泛應用于室內(nèi)裝飾的花卉。但是,紅掌在栽培生產(chǎn)過程中,極易受到細菌性葉斑病的侵害,嚴重時造成毀滅性損失,這是國內(nèi)外紅掌生產(chǎn)上的一大障礙。因此,開展抗病基因工程研究、培育抗病品種具有重要理論意義及應用價值。本研究在前人研究的基礎上,就紅掌的再生體系、農(nóng)桿菌介導的遺傳轉(zhuǎn)化體系、抗菌肽基因及其修飾、融合基因及植物表達載體、農(nóng)桿菌轉(zhuǎn)化等方面進行了研究,取得了以下實驗結(jié)果。1、優(yōu)化了紅掌的再生體系。以盆栽紅掌品種SYN-A的愈傷組織為供試材料,以1/2MS培養(yǎng)基為基本培養(yǎng)基,探討了不同生長素(2,4-D、NAA、IBA)、不同細胞分裂素(6-BA、TDZ)及其濃度配比對紅掌愈傷組織不定芽分化的影響。在本試驗條件下,紅掌愈傷組織的不定芽分化率都達到了76.67%以上,其中最優(yōu)培養(yǎng)基組合為1/2MS+6-BA 0.5 mg/L+2,4-D 0.1 mg/L,愈傷組織的不定芽分化率可達100%,再分化系數(shù)高達31.06,且不定芽生長良好。2、探明了紅掌對三種抗生素的敏感性差異。試驗結(jié)果表明,紅掌外植體對潮霉素的敏感性高于卡那霉素,前者更適用于紅掌轉(zhuǎn)基因組織的篩選,其使用濃度以20mg/L為宜;其次,紅掌外植體對頭孢霉素的敏感度較低,在添加濃度為600 mg/L、培養(yǎng)8周時,外植體依然能正常生長,試驗時使用濃度500 mg/L即可達到良好的抑菌效果。3、初步建立了農(nóng)桿菌介導的紅掌遺傳轉(zhuǎn)化體系。以紅掌愈傷組織為受體材料,以農(nóng)桿菌EHA105為供試菌種,通過正交試驗方法,探討了外植體預培養(yǎng)時間、農(nóng)桿菌侵染時間、超聲波處理時間和共培養(yǎng)時間等對遺傳轉(zhuǎn)化的影響,其影響程度依次為共培養(yǎng)時間預培養(yǎng)時間侵染時間超聲時間。初步建立了適用于紅掌的遺傳轉(zhuǎn)化體系:切取1 cm×1 cm×0.5 cm大小的愈傷組織塊、預培養(yǎng)3 d,以OD600值為0.6的菌液、侵染時間20 min,侵染期間超聲波處理20 s,無需共培養(yǎng),而后經(jīng)抑菌培養(yǎng)、篩選培養(yǎng),可獲得抗性愈傷組織。4、構(gòu)建了含抗菌肽融合基因的植物表達載體及工程農(nóng)桿菌。應用生物信息學方法,以病程相關蛋白PR1a的信號肽與抗菌肽Shiva-1為基本序列,利用紅掌偏愛密碼子進行修飾優(yōu)化后,合成了含特異酶切位點的抗菌肽融合基因(命名為Aa AMP),并連接入中間載體p CAMBIA1301,成功構(gòu)建了植物表達載體p CAMBIA1301-Aa AMP,進而轉(zhuǎn)化農(nóng)桿菌EHA105獲得了工程菌株。
[Abstract]:Anthurium (Anthurium andraeanum) for Araceae Anthurium perennial herbaceous flowers, flowers and leaves because of its peculiar shape, long flowering, often used as cut flower material; potted plant types is exquisite, is widely used in indoor decoration flower. However, in the cultivation of Anthurium production process, vulnerable to bacterial leaf spot damage, serious when a devastating loss, which is home to a large obstacle on Anthurium production. Therefore, it is of great theoretical significance and application value to carry out the research on the genetic engineering of resistance to disease and to cultivate the disease resistant varieties. This study on the basis of previous research on Anthurium regeneration system and Agrobacterium mediated genetic transformation system, antimicrobial peptide gene and its modification, fusion gene and plant expression vector and Agrobacterium transformation and other aspects of the study, obtained the following results. 1, optimize the regeneration system of anthurium. The potted anthurium callus variety SYN-A as the tested material, with 1/2MS as basic medium, effects of different auxin (2,4-D, NAA, IBA) and cytokinins (6-BA, TDZ) of different concentration ratio and its influence on Anthurium callus differentiation of adventitious buds. In this experiment, the callus differentiation rate of adventitious buds have reached more than 76.67%, the optimal medium was 1/2MS+6-BA 0.5 mg/L+2,4-D 0.1 mg/L combination, the callus differentiation rate of adventitious buds was 100%, and the differentiation coefficient was 31.06, and the growth of adventitious buds were good. 2, proved the difference in sensitivity to three antibiotics of anthurium. The test results show that Anthurium andraeanum explants of hygromycin sensitivity is higher than that of kanamycin screening, the former is more suitable for the use of transgenic Anthurium tissue, the concentration should be 20mg/L; secondly, on Anthurium andraeanum explants cephalosporin sensitivity in low concentration was 600 mg/L and cultured for 8 weeks. The explants still can grow normally, the test using concentration of 500 mg/L can achieve good antibacterial effect. 3, preliminary established Anthurium Agrobacterium mediated genetic transformation system. With Anthurium calli, Agrobacterium tumefaciens EHA105 as tested strains, through orthogonal test method, discusses the explants culture time, Agrobacterium infection time, ultrasonic treatment time and incubation time of genetic transformation, the degree of CO cultivation time of pre culture time infection time of ultrasonic time. Establishment of genetic transformation system for Anthurium: cut 1 cm * 1 cm * 0.5 cm in size of callus block, pre cultured for 3 D, the OD600 value is 0.6, the bacteria infection time 20 min infection during ultrasonic treatment for 20 s, there is no need of culture, and then by bacteriostatic culture, Shai Xuanpei raise, can obtain the resistant callus. 4. The plant expression vector and Agrobacterium tumefaciens containing the antibacterial peptide fusion gene were constructed. Using bioinformatics method, and the antibacterial peptide Shiva-1 signal peptide pathogenesis related protein PR1a as the basic sequence, modified by optimization of codon preference of Anthurium, antibacterial peptide fusion gene containing specific restriction sites were synthesized (named Aa AMP), and connected into the intermediate carrier P CAMBIA1301, successfully constructed plant the expression vector p CAMBIA1301-Aa AMP, and then transformed into Agrobacterium EHA105 strain to obtain the engineering.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S682.14

【參考文獻】

相關期刊論文 前10條

1 易懋升,黎揚輝,張志勝,劉琳,蘭天維,夏晴,譚玉鳳;紅掌種質(zhì)資源數(shù)據(jù)庫的建立[J];廣東農(nóng)業(yè)科學;2005年01期

2 韓美麗;陸榮生;吳耀軍;杜曉莉;;農(nóng)桿菌介導的抗菌肽A基因轉(zhuǎn)入沙田柚影響因素研究[J];廣西植物;2006年05期

3 余云舟,杜娟,王罡,季靜;重組質(zhì)粒導入根癌農(nóng)桿菌凍融法的研究[J];吉林農(nóng)業(yè)大學學報;2003年03期

4 杜平;邵小斌;;我國紅掌的研究進展[J];江蘇農(nóng)業(yè)科學;2011年06期

5 王可洲,李浩,劉學玲,朱鍵,馮墨竹,舒心;抗菌肽的分類及作用機理研究進展[J];科學技術(shù)與工程;2005年07期

6 薛菲;孫春玉;蔣世翠;王康宇;于洋;王義;張美萍;;植物轉(zhuǎn)基因技術(shù)及其應用[J];吉林蔬菜;2014年05期

7 趙海紅;張曉申;王慧瑜;李曉青;王曉云;;紅掌組織培養(yǎng)再生體系建立的研究[J];農(nóng)業(yè)科技通訊;2014年02期

8 夏春華;世界紅掌切花業(yè)概況和發(fā)展海南紅掌切花生產(chǎn)的思路[J];熱帶農(nóng)業(yè)科學;2001年01期

9 魏文輝,宋運淳,覃瑞;植物同源異型基因及同源異型盒基因的研究進展[J];生物化學與生物物理進展;2000年02期

10 張海波;金莉莉;王秋雨;;抗菌肽活性及天然抗菌肽的改造[J];生命的化學;2011年02期

相關碩士學位論文 前3條

1 孟鶴;花燭屬親緣關系分析與細菌性枯萎病的檢測[D];中國農(nóng)業(yè)科學院;2011年

2 李小軍;農(nóng)桿菌介導NPR1基因轉(zhuǎn)化安祖花的研究[D];上海師范大學;2005年

3 劉p，

本文編號：1345701