Preliminary Research Of Mutation Mechanism Of Silkworm Fibroin Mutant Nd~x

Silk gland is the only endocrine gland with the capability of silk synthesis and secretion, which is the strongest gland for protein synthesis among insects.Thus,it is the biological basis for silk industry and provide with important economic value.Silk gland can be morphologically and functionally divided into 3 parts,which are anterior silk gland(ASG),middle silk gland(MSG) and posterior silk gland(PSG).ASG is the pipeline for liquid silk protein,whereas,MSG and PSG are sites for efficient protein(fibroin and sericin) synthesis.Sericin is synthesized in MSG while fibroin is synthesized in PSG.Meanwhile,silk gland is an endoradiosonde organism,there is no mitosis or karyokinesis in the silk gland cell,but the genome replication is still in progress for several times without normal cell mitosis.In fact,the cell cycle is the alternation of G1 and S phase.Nd^x(serial No.02-030) is one kind of silk mutant that preserved by the gene library of our institute.The mutant lead to silk gland degradation and about half of them fail to spin.According to the linkage analysis,the Nd^x is located in the 25^th chromosome,which also contains the fibrin heavy chain gene.In this research,we choose Nd-s(serial No.02-020) and Dazao(p50) as control to anatomize their silk glands in different developmental stages,to take count of the silk gland cell, and to observe its growth after DAPI.The results showed that middle or posterior silk gland(MSG, PSG) cell number of 3 different species does not reveal a distinct diversity.In the early 5^th instar, the PSG of Nd-s apparently grows slower than the other 2 controls,especially in length that is only half of the controls.While mounting,the length is only one-forth of the controls.But the MSGs of 3 samples are nearly the same ostensibly.According to the Nuclear Staining obsevation,in the 5^th instar the nucleus of MSG of 02-030 mutant exhibits a dendrite propagation,just the same as the controls,while the nucleus of PSG only expand lengthways,which different from the natural nucleus of silk gland cell.So we believe that the degradation is not a decrease in cell number or atrophy in process of growth,but is a shortness of PSG caused by a heteroplasia in the cleavage of nucleus.We speculate the gene is associated with cell division.Bioinformatics analysis of the silkworm No.25 chromsome where the Nd^x located on showed that the NO.25 chromsome contains 436 genes,of which 364 protein-coding genes have been annotations.72 genes are found to have no similarity with reported genes among species,so they are probably unknown genes or pseudogenes.GO category assay of the 364 annotated genes showed and they are involved in cell components,organelle binding activity,catalytic activity, physiological regulation,cellular processes,metabolic processes and pigmentation,however,most of them are focused on the binding activity,catalytic activity and metabolic function.Normalized microarray data was filtered with the threshold of signal intensity 300.Nine silk gland specific gene probes from No.25 chromsome are found(signal intensity＞300),3 genes highly expressed in silkgland(10 times of those in other tissues) are also detected in other tissues(signal intensity＞300).6 of them are important silk gland specific genes involve in juvenile hormone regulation,fibroin H chain transcription regulation and intracellular transport,etc.At the same time,6 juvenile hormone esterase genes are found in the No.25 chromosome,two of them only express in silk gland,especially the MSG.Nd(2) and Nd^x are both allelic mutant species,previous researches show that the mutation is possibly located on the disulfide bond in the C-termal of fib-H.In this study,sequencing result of the fib-H C-termal among different strains revealed that the fib-H C-termal sequence of Nd^x is exactly the same as the sequence of the normal strain Dazao.Meanwhile,a transcriptional factor Bmsage that is adjacent to fib-H displays no sequence distinction in CDS between Nd^x and Dazao, as well as SGF-1,and their expression level are nearly the same according to the semi-quantitative RT-PCR.So we believe that Bmsage and SGF-1 are unlikely associated with the Nd^x mutation.In accordance with this study,we proved that it is a false presumption that the Nd mutation is cause by the disulfide bond in the C-termal of fib-H.Further studies are still need to be performed to find out the real cause of the Nd mutation.