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caitikuan

ľ³æ (СÓÐÃûÆø)

According to the pore-formation model, the binding of protease-activated toxin to cadherin is essential for the removal of helix a-1, which in turn promotes oligomerization.
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Therefore, we hypothesized that modified Cry1Ab and Cry1Ac toxins lacking helix a-1 (referred to hereafter as Cry1AbMod and Cry1AcMod) could form oligomers without cadherin.
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To test this hypothesis, we compared oligomerization of native and modified Cry1Ab and Cry1Ac in the presence and absence of toxin-binding cadherin fragments.
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Toxins were activated by trypsin to mimic the protease activation that occurs in the insect gut.
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Previous work shows that trypsin-activated Cry1Ab forms 250-kD oligomers in the presence of Manduca sexta cadherin fragments containing toxin-binding regions corresponding to cadherin repeats 7 and 11 or a singlechain antibody (scFv73) that mimics these cadherin toxin-binding regions.
ÒÔÍùµÄ¹¤×÷±íÃ÷£¬´æÔÚÑ̲ÝÌì¶êÖеÄÒȵ°°×ø¼¤»îCry1Ab250 kDµÍ¾ÛÎï½á¹¹µÄ¸ÆÕ³µ°°×Ƭ¶Îº¬Óж¾ËؽáºÏÇøÓò£¬¸ÃÇøÓòÏàÓ¦µÄ¸ÆÕ³µ°°×ÖØ¸´7ºÍ11»òµ¥Á´¿¹Ì壨 scFv73 £©¡£¸Ã¿¹ÌåÄ£·ÂÕâЩ¸ÆÕ³µ°°×¶¾ËؽáºÏÇøÓò¡£
We found that trypsin activated Cry1Ab and Cry1Ac formed oligomers in the presence of a protein fragment corresponding to cadherin repeat 12 (CADR12), an important toxin-binding region (21), but not without cadherin or with cadherin repeat 9 (CADR9), a region that does not bind toxin.
ÎÒÃÇ·¢ÏÖ£¬Ñ̲ÝÌì¶êÌåÄÚÒȵ°°×ø¼¤»îCry1AbºÍCry1AcÐγɵ;ÛÎïÊÇÔÚÏàÓ¦µÄ¸ÆÕ³µ°°×ÖØ¸´12 £¨ CADR12 £©µ°°×Ƭ¶Î´æÔÚµÄÇé¿öÏ·¢Éú¡£CADR12£¬Ò»¸öÖØÒªµÄ¶¾ËؽáºÏÇø£¨ 21 £© £¬µ«²¢·ÇûÓиÆÕ³µ°°×»ò¸ÆÕ³×ŵ°°×ÖØ¸´9 £¨CADR9 £©£¬Ò»¸ö²»º¬¶¾ËؽáºÏµÄÇøÓò¡£
In contrast, trypsin-activated Cry1AbMod and Cry1AcMod formed oligomers without cadherin, although not as efficiently as the wild type with CADR12.
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11Â¥2009-01-15 13:27:32
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caitikuan

ľ³æ (СÓÐÃûÆø)

We used RNA interference (RNAi) to reduce production of cadherin protein (Fig. 2 and fig. S1) and decrease the susceptibility of M. sexta larvae to Cry1Ab.
ÎÒÃÇʹÓõÄRNA¸ÉÉæ£¨ RNAi £©£¬ÒÔ¼õÉÙ²úÉúµÄ¸ÆÕ³µ°°×£¨Í¼2ºÍͼS1£©ºÍ½µµÍÑ̲ÝÌì¶êÓ׳æ¶ÔCry1AbµÄ Ò׸ÐÐÔ¡£
Larvae injected with either 1 mg of cadherin double-stranded RNA (dsRNA) or water only (control) ate a diet treated with 20 ng of Cry1Ab protoxin/cm2.
Ó׳æ×¢Éä1ºÁ¿ËµÄ¸ÆÕ³µ°°×µÄË«Á´RNA »òË®£¨¶ÔÕÕ£©£¬Ê³Îï²ÉÓà 20Äɿ˵ÄCry1Ab protoxin/cm2 ÒûʳÖÎÁÆ¡£
After 3 days, survival was 92% for 48 RNAi-treated larvae versus 0% for 48 control larvae (chi-square test, P < 0.001).
3Ììºó£¬48 Ö»RNA¸ÉÈÅÖÎÁƵÄÓ×³æ´æ»î92 £¥£¬¶ÔÕÕµÄ48Ö»´æ»îÂÊΪ0 £¥£¨¿¨·½¼ìÑ飬 P <  0.001 £© ¡£
The next experiment showed that cadherin silenced M. sexta larvae were much more susceptible to Cry1AbMod than to Cry1Ab (fig. S2).
½Ó×ŵÄÊÔÑé±íÃ÷£¬¸ÆÕ³µ°°×³ÁĬµÄÌì¶êÓ׳棬¶ÔCry1AbMod±È¶ÔCry1Ab¸üÃô¸Ð £¨Í¼S2£©¡£
Confirming the results described above, the survival of larvae fed a diet treated with 20 ng of Cry1Ab protoxin/cm2 was higher for 48 RNAi-treated larvae (92%) than for 48 control larvae (2%)(chi-squaretest, P < 0.001).
ÉÏÊö½á¹û֤ʵÁË£¬Ó׳æÎ¹Ê³ÒûʳÖÎÁÆ20Äɿ˵ÄCry1Ab protoxin/cm2µÄÉú´æ48Ö» RNA¸ÉÈÅÖÎÁƵÄÓ׳棨92 £¥£©¸ßÓÚ48Ö»¶ÔÕÕÓ׳棨2 £¥£© £¨¿¨·½¼ì p<0.001 £©¡£
However, on a diet with 5 ng of Cry1AbMod protoxin/cm2, 48 RNAi-treated larvae had only 2% survival, which is significantly lower than the aforementioned 92% survival of 48 RNAitreated larvae fed a diet treated with 20 ng of Cry1Ab protoxin/cm2 (chi-square test, P < 0.001).
È»¶ø£¬ÔÚιʳ5Äɿ˵ÄCry1AbMod protoxin/cm2 ºó£¬48 Ö»RNA¸ÉÈÅÖÎÁƵÄÓ׳æÖ»ÓÐ2 £¥µÄÉú´æ£¬ÕâÊÇÏÔ×ŵÍÓÚÉÏÊö48Ö» RNAitreatedÓ׳æÎ¹Ê³ÒûʳÖÎÁÆ20Äɿ˵ÄCry1Ab protoxin /ƽ·½ÀåÃ×µÄ92 £¥´æ»î£¨¿¨·½¼ìÑ飬 P < 0.001 £© ¡£

ÎÒÃÇ»¹½øÐÐÍ»±äºÍÒ°ÉúÐÍCry1A¶¾ËضÔÓ׳æµÄ¿¹ÐÔÆ·Ïµ£¨ AZP - R £©ºÍÃô¸ÐƷϵ£¨APHIS-S£©µÄÃÞ»¨º¦³æºìÁ峿£¨ÃÞºìÁ峿£©µÄÊÔÑé¡£ÔÚº¬BtÃÞÉú²úCry1AcÊÔÑéÖп¹ÐÔÆ·ÏµÓ׳æÐÒ´æ¶ø·ÇÃô¸ÐƷϵÓ׳档ÔÚAZP - RÖп¹Cry1AcÊÇÓë¸ÆÕ³ËØÊÜÌå»ùÒòȱʧͻ±äÏà¹Ø¡£¿¹ÐÔÆ·ÏµÖÐCry1AbModºÍCry1AcMod¼õÈõ»òµÖ¿¹¿¹ÐÔ£¨Í¼3ºÍ±í1£©¡£ÔÚ´Ë»ù´¡ÉÏÔì³É50 £¥£¨°ëÖÂËÀŨ¶È£©ËÀÍöµÄŨ¶È£¬AZP - RÏà¶ÔAPHIS-S£¬ AZP - R±ÈAPHIS-S¿¹Cry1Ab>910±¶ºÍ ¿¹Cry1Ac> 3700±¶£¨±í1£©¡£Óë´ËÏà·´£¬AZP - RÖ»ÓÐ2.8±¶ÄÍCry1AbMod £¬¶ø²»¿¹Cry1AcMod £¨±í1£©¡£¶ÔÄÍÒ©ÐÔÓ׳æµÄLC50Cry1Ac±ÈCry1AbMod»òCry1AcMod ¸ßΪ00±¶ÒÔÉÏ£¨±í1£©¡£Ïà·´£¬¶ÔÃô¸ÐµÄÓ׳棬ÌìÈ»¶¾ËØÓÐЧÐÔ³¬¹ýÁË¸Ä±ä¶¾ËØ¡£ÕâÒâζ×Å£¬Ïà¶ÔÓÚÌìÈ»¶¾ËØ×÷ÓÃÓÚÃô¸ÐÓ׳棬ÐÞÊζ¾ËØÔÚÖг¦Îȶ¨ÐÔ½µµÍ£¬µÍ¾ÛÎïÐγÉÄÜÁ¦½µµÍ£¨Í¼1 £© £¬»ò¼õÉٵ;ÛÎïµÄÄÜÁ¦£¬×îÖÕµ¼Ö¶¾ËØÎÞ×÷Óá£

[ Last edited by caitikuan on 2009-1-15 at 14:42 ]
12Â¥2009-01-15 13:45:37
ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû

caitikuan

ľ³æ (СÓÐÃûÆø)

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xu_0501(½ð±Ò+300,VIP+0):thank you very much
The results suggest that in two species of Lepidoptera, cadherin receptor
protein in the larval midgut mediates the toxicity of Cry1A toxins by
facilitating removal of helix a-1, which promotes toxin oligomerization.
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The modified toxins Cry1AbMod and Cry1AcMod lacking helix ¦Á-1 formed oligomers  in vitro without cadherin, whereas native Cry1Ab and Cry1Ac did not.
Ð޸ĺóµÄ¶¾ËØCry1AbModºÍCry1AcModȱ·¦¦Á - 1 ÂÝÐý£¬ÔÚÌåÍâÐγɵ;ÛÎïÎÞ¸ÆÕ³µ°°×£¬¶øÌìÈ»Cry1AbºÍCry1AcÔÚÌåÍâÐγɵ;ÛÎïÓиÆÕ³µ°°×¡£
The modified toxins killed insects with greatly reduced susceptibility to
native Cry1A toxins caused by RNAi silencing of the cadherin gene or by mutations in the cadherin gene.
Ð޸ĺóµÄ¶¾ËØÉ±ËÀÀ¥³æµÄͬʱ´ó´ó½µµÍÌìÈ»Cry1A¶¾ËظÐÐÔ£¬ÕâÖÖ½µµÍÊÇÓÉÀ¥³æÊÊÓ¦ÐÔµÄRNAi³ÁĬ»ò»ùÒòÍ»±ä¸ÆÕ³µ°°×µÄ»ùÒò¡£
These results support the pore-formation model (15) and not the signaling
model, which does not include removal of helix a-1 or toxin oligomerization
.
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If the results seen here with the pink bollworm extend to other lepidopterans,Cry1AbMod and Cry1AcMod could be broadly useful for countering or delaying pest resistance to Cry1A toxins.
Èç¹ûÔÚÕâÀï¿´µ½µÄ½á¹û£¬ÄÜ´ÓºìÁ峿À©Õ¹µ½ÆäËûÁÛ³áÀàÀ¥³æ £¬Cry1AbModºÍCry1AcMod´óÖ¿ÉÓÃÓÚ½â¾ö»òÍÆ³Ùº¦³æ¶ÔCry1A¶¾ËØ¿¹Ò©ÐÔ¡£
However, we do not know whether Cry1AMod toxins kill insects with mechanisms of resistance unrelated to cadherin, such as the disruption of other receptors or decreased protease activation.
È»¶ø£¬ÎÒÃDz»ÖªµÀCry1AMod¶¾ËØÉ±ËÀº¦³æµÄ¿¹ÐÔ»úÖÆÊÇ·ñºÍ¸ÆÕ³µ°°×Î޹أ¬ÈçÆäËûÊÜÌåµ°°×ø¼¤»îµÄÖжϻò¼õÉÙ¡£
Many Bt toxins have structural topology similar to Cry1A, form oligomers, and
induce pores, suggesting that they share a similar mode of action.
Bt¶¾ËغÍCry1AÓÐÐí¶àÏàËÆÈçÍØÆË½á¹¹ £¬ÔÚÐγɵ;ÛÎï¼°ÓÕµ¼¿Õ϶µÄ¹ý³Ì£¬Õâ±íÃ÷ËüÃǾßÓÐÀàËÆµÄ×÷Ó÷½Ê½¡£
It remains to be determined whether, parallel to results with Cry1A toxins,
other Cry toxins lacking helix a-1 can kill resistant insects that have altered
receptors.
Cry1A¶¾ËØÓëÆäËûȱ·¦¦Á-1ÂÝÐýµÄÄܹ»É±ËÀ¸Ä±äÊÜÌåÇÒ¾ßÓп¹ÐÔµÄÀ¥³æµÄCry¶¾Ëؽá¹ûÊÇ·ñƽÐл¹ÓдýÈ·¶¨¡£
In addition, insects can probably evolve resistance to modified Bt toxins
lacking helix a-1.
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Nonetheless, along with native Bt toxins such as Cry2 and Vip3 that have not been used as extensively as Cry1A toxins, the modified toxins broaden the options for pest control.
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[ Last edited by caitikuan on 2009-1-15 at 15:35 ]
13Â¥2009-01-15 14:00:36
ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû

sixmonths

Ìú¸Ëľ³æ (ÖøÃûдÊÖ)

Çå¸èÒ»ÇúÔÂÈç˪

ÒýÓûØÌû:
Originally posted by caitikuan at 2009-1-15 14:00:
The results suggest that in two species of Lepidoptera, cadherin receptor
protein in the larval midgut mediates the toxicity of Cry1A toxins by
facilitating removal of helix a-1, which promotes ...

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14Â¥2009-01-15 21:04:45
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