| The14-3-3proteins, a highly evolutionarily conserved and ubiquitous proteinfamily in eukaryotic cells, have a range of biological functions including regulation ofsignal transduction, stress response, apoptosis, and controlling of the cell cycle. Toinvestigate the function of14-3-3in Spodoptera litura, the full length of14-3-3ζ, namedas SpL14-3-3ζ, was cloned from S. litura on the basis of an expressed sequence tag(EST) of14-3-3ζ from the S. litura fat body suppression subtractive hybridizationlibrary (SSH). By qPCR and RNAi, we studied the function of SpL14-3-3ζ in thedevelopment of S. litura, and the molecular response relationship upon Nomuraea rileyichallenge. Our results will be helpful for understanding the molecular responsemechanism of S. litura to N. rileyi infection, providing a scientific basis for controllingS. litura with the14-3-3protein as the target.①Based on an EST of14-3-3ζ from the S. litura SSH library, the full length of14-3-3ζ was cloned from S. litura using SMART RACE and named SpL14-3-3ζ, whoseamino acids sequence was identified by ORF Finder. The physical and chemicalproperties and the structure of the protein were predicted by Expasy etc. softwares. Thehomologous comparison of14-3-3proteins from various species was being done usingDNAMAN. And then multiple alignment analysis and the phylogenetic tree wereperformed by ClustalW and MEGA software. The results showed that SpL14-3-3ζcDNA was1196bp long with an open reading frame744bp encoding247amino acids.The molecular weight of the protein was28.0kDa with isoelectric point of4.6, withoutsignal peptide or transmembrane domain. Multiple alignment analysis revealed theputative amino acids shared>80%homology with14-3-3ζ from other organisms.Phylogenetic analysis confirmed SpL14-3-3ζ was closely related to other availablelepidopteran14-3-3ζ. Genome structure showed that SpL14-3-3ζ gene contained fourexons, three introns.②Expression profile ofSpL14-3-3ζ during S. litura metamorphosis was analyzedby qPCR. And the results indicated SpL14-3-3ζ was expressed throughout thedevelopmental stages of S. litura. During the larval development,4thinstar larvaeshowed a higher expression with4.44-fold compared to2ndinstar larvae, and the lowestlevel of expression occurred in5thinstar larvae. While during the development of pupa,the highest level of expression occured at prepupa, which was22.8-fold compared to2nd instar larvae.③Spatial expression patterns ofSpL14-3-3ζ in S. litura was analyzed by qPCR.And the results indicated SpL14-3-3ζ expressed constitutively in all examinedtissues/organs, with relatively high expression levels in hemocytes and midgut, whichwere>22.4-and6.2-fold greater compared to in head, respectively, whereas theexpression level in cuticle was relatively low. However, after N. rileyi-challenged for24h, SpL14-3-3ζ expression level could be up-regulated in certain tissues. Compared toeach normal tissue, SpL14-3-3ζ expression levels were enhanced in hemocytes, head, fatbody and midgut by6.5-,3.7-,2.0-and2.8-fold, respectively.④During the time course studied in three indicated tissues, afterN. rileyi infection,SpL14-3-3ζ expression level in hemocytes was increased and occurred the maximalinduction at12h post-infection (15.5-fold compared to0h) and then declined gradually.In fat body, the trend of SpL14-3-3ζ expression level was nearly the same withhemocytes, but reached only half expression level compared to hemocytes at the12htime point (7.7-fold compared to0h). By contrast, SpL14-3-3ζ expression in midgut wasincreased to a peak at6h, and later decreased to the original level (5.6-fold compared to0h).⑤Knocking downSpL14-3-3ζ transcripts by RNAi,100%of larvae could moltand enter into pupate, but compared to control group,55+2%pupa could not emerge toadult and35+3%pupa emerged into malformed adults. Besides, after silencing ofSpL14-3-3ζ expression, significantly increased S. litura sensitivity to fungal infection,and resulted in a higher mortality rate of S. litura during the larval development.Conclusion:14-3-3ζ gene was first cloned from S. litura, named as SpL14-3-3ζ, and itwas expressed continuously but unevenly during the larval developmental stages and indifferent tested tissues. The expression can be induced by N. rileyi infection and higherlevels of expression appeared in the tissues involving in insect immunity. Afterinterference, larvae could not grow normally, and had higher mortality and lower LT50after N. rileyi infection. These results will provide novel insights into the14-3-3ζ signalregulation which may be related to host defense as well as larval development in S.litura. |
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