ABSTRACT

RNAi approach was attempted to impart resistance against Banana bract mosaic virus (BBrMV). An intron hairpin RNA (ihpRNA) vector targeting its replicase gene was constructed in pSTARLING vector. A fragment (421 bp) of the BBrMV replicase gene isolated from the infected sucker of banana cv. Nendran (Musa AAB) was used for making ihpRNA cassette. The ihpRNA cassette (NotI fragment) was mobilised to the binary vector pART27 in Agrobacterium tumefaciens strain LBA 4404 for transformation. Embryogenic calli derived from the immature male inflorescence of cv. Nendran maintained in MS medium supplemented with BA 0.1 mg L⁻¹ and picloram 1 mg L⁻¹ were transformed with LBA 4404. Transformed calli were subjected to embryo induction in semisolid MS medium supplemented with BA 2 mg L⁻¹ and IAA 0.5 mg L⁻¹ and germinated on half-strength MS medium with BA 2 mg L⁻¹ and IAA 0.5 mg L⁻¹, with a maximum regeneration of 25% under the selection pressure of 100 mg L⁻¹ kanamycin. The regenerated shoots were confirmed for the presence of ihpRNA construct by PCR with primers specific for sense-intron-antisense fragment, npt II and cre intron. The transgenics challenged with BBrMV showed no infection. Untransformed plants showed infection and developed disease symptoms after 3 months.

摘要

尝试使用RNAi方法赋予对香蕉片花叶病毒（BBrMV）的抗性。在pSTARLING载体中构建了靶向其复制酶基因的内含子发夹RNA（ihpRNA）载体。从感染的香蕉cv吸盘中分离出的BBrMV复制酶基因的一个片段（421 bp）。Nendran（Musa AAB）用于制备ihpRNA盒。将ihpRNA盒（Not I片段）动员到根癌农杆菌LBA 4404中的二元载体pART27进行转化。胚性愈伤组织来源于不成熟的雄性花序。Nendran维持在补充有BA 0.1 mg L ^-1和Picloram 1 mg L ^-1的MS培养基中用LBA 4404转化的愈伤组织转化进行胚胎诱导在补充有2 BA毫克的L半固体MS培养基^-1和IAA 0.5毫克的L ^-1和发芽半强度MS培养基上以BA 2毫克的L ^-1和IAA 0.5 mg L ^-1，在100 mg L ^-1卡那霉素的选择压力下，最大再生率为25％。通过PCR，用对正义-内含子-反义片段，npt II和cre内含子具有特异性的引物，证实了再生的芽中存在ihpRNA构建体。用BBrMV攻击的转基因未显示感染。未转化的植物在3个月后显示出感染并出现了疾病症状。