向日葵黄萎病菌突变体库的建立及其阳性转化子的鉴定

The establishment of T-DNA library and the characterization of positive transformants of sunflower Verticillium dahliae

为揭示向日葵大丽轮枝菌Verticillium dahliae Kleb.的致病机理,利用农杆菌介导法将带有潮霉素抗性标记和绿色荧光蛋白报告基因的双元载体转入大丽轮枝菌的分生孢子中并获得阳性转化子,以野生型菌株为对照,对随机挑取的阳性转化子的菌落形态、菌丝生长速率、产孢量、粗毒素分泌量和致病力进行了研究。结果表明,共获得800株阳性转化子,随机选取的40株阳性转化子中有2株的菌落只产生白色气生菌丝,不能形成黑色微菌核。与对照相比,40株转化子的生长速率均有不同程度降低,其中转化子A1生长速度降低最显著,菌落直径仅为3.28 cm,比对照下降了38.92%。40株转化子中有3株的产孢量高于对照,其中转化子A9的产孢量最高,为3.50×10~7个/mL,比对照提高1.10倍;转化子A1的产孢量最低,仅为1.35×10~7个/mL,比对照下降了19.16%。40株转化子中有4株的粗毒素分泌量较对照显著升高,占测定菌株的10%,有24株较对照显著降低,占60%,其余12株与对照无显著差异。40株转化子中有3株的致病力较对照显著增强,占测定菌株的7.5%;有7株的致病力较对照显著降低,占17.5%;其余30株与对照无显著差异。

To unravel the pathogenic mechanism of sunflower Verticillium dahliae Kleb., a binary vector contained hygromycin resistance gene and green fluorescent protein gene （GFP） was transformed in- to the conidia of If. dahlia using Agrobacterium tumefaciens-mediated transformation system （ATMT） and 800 positive transformants were obtained. Forty out of 800 transformants were chosen randomly to compare the colony morphology, growth rate, conidia production, toxic production and pathogenicity. The results showed only two transformants produced white aerial hyphae and no microsclerotia formed. Compared with wild type, all the tested positive transformants showed the reduced growth rate, and A1 mutant was the most slowest one, the colony diameter was only 3.28 cm, and the reduce rate was 38.92%. Three transformants showed higher conidia production ability than the wild type. Among them, A9 mutant showed the highest conidia production ability with 3.50×10^7 conidia/mL, 1.10 times as the wild type; A1 mutant was the lowest one, the conidia production was only 1.35× 10^7 conidia/mL, and the decreased rate was 19.16%. Regarding to toxin production ability, 24 transformants secreted less toxin than wild type strain, accounted for 60% of the tested transformants and only 10% transformants showed the increased tendency. The pathogenicity assay was also performed via roots dipping system, three transformants showed increased tendency, whereas, seven transformants showed decreased tenden cy, 30 transformants showed the same pathogenicity as wild type strain.