摘要
本研究对水稻和油菜进行内生菌的分离,各自分离到了66和63株细菌,其中杆菌、革兰氏阳性菌,有芽胞的占优势。SJ-10和YY-23分别为水稻和油菜内生菌,经生理生化、16SrRNA序列鉴定及构建16SrRNA系统进化树,YY-23鉴定为苏云金芽胞杆菌,SJ-10属于肠杆菌属。WB800为枯草芽胞杆菌。以灌根,浸种及接种无菌苗等方法回接水稻和油菜,定殖回收结果表明SJ-10、WB800和YY-23能在水稻和油菜体内长期定殖。且三株菌均可在小麦、玉米、油菜、红菜苔、萝卜等多种植物体内定殖,SJ-10和WB800在三个月后仍可以从上述植物中分离到,YY-23在两个月后可以分离到。菌液浸种24h后播种,20天后,经SJ-10浸种后油菜鲜重比对照增加了52.5%,经YY-23浸种后油菜的鲜重比对照增加了113.5%。经WB800浸种30天后水稻的鲜重比对照增加了62.16%。
利用半夏凝集素(PTA)构建分泌表达穿梭载体,转入水稻内生菌SJ-10和WB800中,进行SDS-PAGE及Western Bloting分析,SJ-10转化子的菌体蛋白和WB800转化子上清的中可以检测到PTA,两菌进行抗虫实验分析结果表明PTA可以明显的抑制白背飞虱的群体增长量。19天时群体数量达到最大值,此时对照的白背飞虱的群体数量是SJ-10-PTA的3.38倍,是WB800-PTA的98.7倍。到26天时CK与WB800-PTA白背飞虱的数量降为0,且CK和SJ-10试验苗在此时全部死亡。WB800、SJ-10-PTA与WB800-PTA试验苗生长正常。
将内生菌SJ-10和YY-23添加到人工营养液中饲喂棉蚜,SJ-10在饲喂第6天时存活率比对照明显降低,差异显著(P<0.05),而YY-23无抗虫活性。
上述结果表明三株内生菌能稳定在植物体内定殖,并具有促生长作用,重组菌SJ-10和WB800有明显的抗白背飞虱活性,野生菌SJ-10同时具有一定的抗蚜虫活性。因此植物内生菌可作为宿主菌,表达抗虫蛋白,用于抗刺吸式口器同翅目害虫。
In this paper,66 and 63 strains were isolated from rice and rape, respectively. The separation results showed that the bacillus, gram-positive bacteria, spores were in the majority. SJ-10, and YY-23, isolated from rice and rape, were identified as Enterobacter sp. and Bacillus thuringiensi, respectively, based on the basic properties tests,16SrRNA and 16SrRNA phylogenetic tree. WB800 was Bacillus subtilis. The three strains were able to colonize in rice and rape by dipping the seeds, watering the roots, and inoculating aseptic seedlings. The inoculation treatment had been proved that they could live in many plants, such as wheat, maize, rape, chinese kale, navet. After three mothes, SJ-10 and WB800 could be colonized from the plants mentioned above. After two mothes, YY-23 also could be colonized. Rape seeds were dipped for 24h with YY-23 and SJ-10, then sowed. After 20 days, the fresh weight of rape plants was increased by 113.5% and 52.5% respectively. Rice seeds were dipped like rape with WB800, and after 30 days, the fresh weight of rice was increased by 62.16%.
A shuttle and secreting expression vector including Pinellia ternate agglutinin (PTA) gene was constructed and introduced into endophytic SJ-10 and WB800. SDS-PAGE and Western Bloting analysis showed that PTA protein could be detected in recombinant SJ-10 and WB800. After inoculated rice seedlings with the recombinant endophytic bacteria, insecticidal activity against white backed planthopper was detected. The results showed that the PTA protein expressed by the recombinant SJ-10 and WB800 could obviously decrease the survival and fecundity of white backed planthopper. The insect number on rice seedlings in control group was 3.38 times of that inoculated with recombinant SJ-10, and 98.7 times of that inoculated with recombinant WB800 at day 19. At the end, rice seedlings in the control group and SJ-10 all died, but seedlings inoculated with WB800, the recombinant WB800 and SJ-10 grown very well.
Cotton aphid was fed with artificial diets adding bacteria, result showed that the survival rate of aphid in SJ-10 group was lower than CK on the sixth day, the difference was very distinctive (p<0.05). However, YY-23 expressed no resistance to aphid.
In a word, the results showed that the three strains could colonize stably and promote growth in plants. The rice seedlings inoculated with the recombinant endophytic bacteria expressing PTA protein were endowed with the anti-pest activity, and could reduce damages caused by white backed planthopper. Wild SJ-10 had insecticidal effect on cotton aphid. Therefore, the endophytic bacteria could be used as a living vector for expressing anti-pest proteins to control sap-sucking Homoptera insects in the future.
引文
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