Negative Regulation Of Bacillus Thuringiensis Nematicidal Cry6Aa Gene Expression, The Ingestion Mode Of Nematicidal Crystal Protein By Root-Knot Nematode

Bacillus thuringiensis(Bt) is a well-known gram-positive and spore-forming soil bacterium that produces parasporal crystal protein during the sporulation phase of its growth cycle.The crystal protein exhibits high toxicity to many insects,so it was extensively used in biocontrol for insects.Recently,it was found that Bt showed high toxicity to nematode.This work studied the activity of Bt to plant-parasitic nematode from the perspective of control efficiency of Bt against root-knot nematode,the expression regulation of nematicidal cry6Aα2 and the action mode of crystal protein against plant-parasitic nematode.1.The nematicidal activity and control effect of Bt strain YBT-1532 against Melodogyne hapla was assayed.The strain harbors cry1Eα6 which encodes a 130 kDa nematicidal crystal protein,and the strain produces abundant thuringiesin which exhibits nematicidal activity.In this work,bioassay of the crystal protein and thuringiensin against 2^nd stage juvenile of M.hapla was carried out.The results indicated that they both exhibited high nematicidal activity with a LC₅₀ value of 19.32μg/mL and 25.87μg/mL,respectively. Based on the results,the control efficiency of YBT-1532 against root-knot nematode in green house and field were assayed.The results showed that strain YBT-1532 could efficiently inhibited infection of M.hapla to tomato in green house,obviously reduced the formation of galls and eggs,and decreased the population of nematode in soil.However, it showed no obvious control efficiency to M.arenria in field assay,but it could increase the yield of peanut about 6%.2.The negative regulation mode of cry6Aα2 expression was primarily studied. Nematicidal crystal protein gene cry6Aα2(orf1) which was followed with orf2 was cloned from Bt strain YBT-1518.The two genes were separated by a stern-loop.The stem-loop inhibited expression of orf2,made it expressed at low level.When the stem-loop was deleted,orf2 coexpressed with orf1 at high level,meanwhile,the expression level of orf1 was decreased remarkably.When orf2 was mutated,the level of orf1 expression was enhanced obviously.EMSA indicated that the ORF2 protein could bind with the inverted-repeat sequence.In conclusion,the stem-loop disturbs orf2 expression;ORF2 bind inverted-repeat sequence,which maybe open the stem-loop,lead to decrease the stability of the mRNA of cry6Aα2,so down-regulates cry6Aα2 expression. In Bt,high level expression of cry genes is due to many positive regulation factors. However,a negative regulation factor was founded in this study.It provided new insight into the regulation of cry genes expression,and is an example of novel negative regulation and a potential method for enhancing the expression level of cry genes.3.The action mode of crystal protein toxin to root-knot nematode was primarily studied. Due to plant-parasitic nematodes parasitizing on plant tissues,uptaking nourishment from host cell through stylet,the crystal protein is difficult to enter into nematode through stylet.However,many experiments showed that crystal protein exhibited toxicity to plant-parasitic nematode.How the crystal protein enter into plant-parasitic nematode and target it is not clear.In this work,Cry6Aa2 was detected from M.hapla treated with Cry6Aa2 by western blot,and the molecular weight of Cry6Aa2 was enhanced obviously, which indicated that Cry6Aa2 was bound with the receptor in nematode;when M.hapla was treated with Cry5B,the protein was degraded into about 70 kDa.The results suggested that the action mode of Cry6A and Cry5B is different.When M.hapla was treated with Cry51Aa1,a non-toxic protein to nematode,the protein was detected from M. hapla,but the molecular weight was not changed.M.hapla was treated with Cry6Aa2 labeled with rhodamine,observation of confocal microscope indicated that fluorescent signal appeared in the cuticle firstly,and then appeared in the lumen of nematode.Finally, the fluorescent signal centralized in the tissue of intestine.The fluorescent signal in stylet was not observed always.This result suggested that Cry6Aa2 maybe enter into nematode through cuticle.