{£9 #94 ACTA AGRONOMICA SINICA 2007,33(5) :697 - 702 up: //www .chinacrops . org/awxb/ ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@ chinajoumnal net HRELEEARREAREND HT LED ERR FRM Res KR HF! (NP Ae 8% BEAN AEE IL 08400071 LF He CB A EEC HE 20082) Be ALFA FEST SPE Ue HEI A SER EF CABG-3A WE a HR Ae HB IC FBV) HA BI ERIS PT, AUREREDEPLAE He PBN BER PCR ARMA AER US 70% . RULER 4 MARIE HRA T, FLEE Southem IE SBA ,3 PAPI A 1 PARI OLA; Northern SPATS UES FON AGDITE PERE ABATE PAs. HARI RA SAAR AM ABS RAMS TSA CUS ARLEN I 4 TR REET 1, RH ITE BEAT ES 1, AUT, ARAL ADRL, EG he RAR TE A IG CT EH Re a ATES EO BE HR TF AE EO INS HD HH A AA AT A IE 118 $F SRB BE HAG 12.3% 6 RON: RACK ACARD HOE TE Analysis of Cotton ( Gossypium hirsutum L.) Plants Transformed with a Silkworm Fibroin Light Chain Gene SHANG-GUAN Xiao-Xia'?, WANG Ling-Jian?, LI Yan-E!, LIANG Yun-Sheng' , and WU Xia! Coton Remarch Inte, Shani Agiculura Academy, Yuncheng 044000, Shamir * lniute of Past Physilogy and Eeology, Shanghal Inatiate for Biologic! Sciences, Chinese Academy of Sciences, Shanghai 200032, China) Abstract: By Agrobacterium-mediated transformation, a silkworm fibroin light chain gene (FBN), driven by a fiber- specific promoter GAE6-3A, was introduced into the upland cotton G. hirsutum L. ev RIS. The plant expression vector used in this study also contains a GUS gene,and a npt Id gene driven by 35S promoter, respectively. Kanamycin-resistance analysis, GUS-histochemical staining, and PCR detection were conducted in the 20 Ty generation plants form 11 different transgenic lines. Seventeen plants showed kanamycin resistance, 13 plants were positive in GUS detection, and 14 were positive by PCR analysis for the silkworm fibroin light chain gene FBN. Thirteen positive plants were obtained by the three ‘examination methods among the 20 Tp generation plants. Souther hybridization of T; progenies of lines HI8, H21, H32, and H34 were performed, the result showed that among the four T; lines, three lines contained two copies and one line contained a single copy of the transgene, no clear hybridization signal was detected for non-transformed control plants, indicating thet FBN gene was inserted into the cotton genome by Agrobacterium-medicated transformation. Northem ‘analysis demonstrated that the FBN gene was indeed expressed in the transgenic cotton fiber of above four transgenic lines Kenamycin-resistance assay and GUS-histochemical staining was used for screening transgenic progenies. Kanamycin assay ‘was performed in the field as a preliminary screening, which may be influenced by climate and other ciroumstance factors, however, the operation is simple and fast, laying the basis for further selection by GUS histochemical localization Proceeded the two methods together, the limitation of each measure can be compensated, accordingly the efficiency of homozygous breeding of transgenic progenies could be increased obviously. Till now four Ty positive lines, in addition to several T; or T; transgenic plants were obtained, and the foreign gene could be inherited stably form generation to generation. Analysis of fiber quality traits showed that, among the six transgenic lines whose cotton fiber quality was evaluated, the fiber strength of three lines were increased, with the highest in transgenic line HI8. These results demonstrate that plant genetic engineering of silkworm fibroin genes has a great potential for improving cotton fiber quality. Keywords: Silkworm fibroin light chain gene; Transgenic cotton; Cotton fiber MARE A-HARAR,—HEGRWRK AXP LHRH. MTRNEKRALES MINA: SEER IE RET A (863 HF-21) 1H (2004AA212102 200344222080) HEART: EAE 1974~), 3 OE EUR SERA I AEM ME EAA. uml appx@ sb ac. on ‘Received( HAN FM): 2006-08-01; Accepted ($3 11 MH): 2006-11-04,698 fe * ff 33 GHEE SRST Dy a" «SL FS BH A ARSE RB BLK IER HE AR A A KB KEPERER LAS EAM HMMS FEE BEI AYE A FAB 8 HE BER eH A SALEM ARTEL CRA PRG EEF BO. John S59 He RE a A PhaB #1 PhaC MS AAR 7E , 19% ME TE PERMA SATE MEM, A BE THR Calgene 2 7) 5 3% ft BGA SEA HATE E18 AOR ARE BBR RUE. Li HEAL ARAFAT ESAS BAMA A OTR ERA BADE BE acs A acsB SHAPE A TRY PEARCTEREAREMH— ERM. ROE PP ACH AB HE Bs ST SBR AY EES A AL PEAT A EN FREAL ERMA MES. RANEERPHRKERAMLR, BE JK HP 3) BT RH tS EG EB ( GenBank AAA2T840) . BEE ah He ch 2 AE ML AL 1 Ae HE 48,4 325 kDs1 HABE 25 kD. RASA RR ANA MARSHY 2 BBS MR YE ERHARR- TAR SARA MAM ERA, SEA ER AY RI RA RA Re BUF AG BP AR IRE STR HI AR Bt BAS TET AE RH A UAL EP RECAEAA RE ORK Aa ARE AE, SME, RAE BK BFSSAEAAIHKH. ALAM RAR SPE Hit ME GAEO-3A'" Jes ob FH ASE ‘$2 8E35 H4(cilk fibroin light chain gene) FBN #¢AMME, ESLER PAE IPRA CASAC BA OTE FEF Dy AE A PP a OP. 1 BHSAE 1.1 8 $e BE 3 AR Es BS A ( L.A RIS. ARSE FP eK oF CARO 3A MRL ASE PBN hh Ae Be bE oe Be DEBESHRARRUATA RH. ARAM 4&2) PCAMBIA2301,& 35S 06 3h 7 9 8h th ABH SERRA pe TB CUS MRA. MEE RRA RL, HAN PRE 1 Phos Gossypium hirsutum Taba Hs BLS He BAR RG DAF BY LBA 04 oh Kanamyen (R) Noo HAN 13336 bp BR Bi RamRNR MANE Fig.1 Sketch map of transgenic vector HAN 1.2 Wik 1.2.1 #RAREORE ARAMA FRM KBB 50 mg LHF 100 mg L' HE MER 300 mg LY YEB AWS FEM LE I~ 3d Jes DA AF PAL AER HY YE HR ARSE IE EP, F WCHARPBRNR. BRT 4 000x g BAD 10 min, TIE A WH 30 g LAT 100 pmol"! #8) 1/2 MS RUSTE RTI, 1H ODay 18404-0624 FABRE. 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Gelrite, pH 6.0; MS $27 KNO, DIfiE, AAR NH,NO,) , S702 BA AE. 1.2.2 AER BARE Hea 1.2.2.1 RABBI RH FARE 16 BAB ERR BR HK HE EH PR IE A ME ALKBoe AO TIURB AT He 7 eR REL HR HB EH YB CIE BRAG RSET, Ph EB AY BREDA. 1.2.2.2 GUS AR RT, RMA A RMIEA FM BMF A 0.25 mg mL‘ X-gluc 9.5 BE ¥K(100 mmol L~' pH 7.0 BEAR TH .50 mmol L** K,[Fe(CN),].50 mmol L”* K,[Fe(CN),] 31,0. 10 mmol L“' EDTA,O.1% Triton X-100) ,37°C He #898 fA 4 bh DAL 70% Z, BE £8, RA © 1.2.2.3 M7 DNA HSE WASSER 7E PCR aye RUE Ot DNA SAP ICMR(12). A FBN 4 5¢ IE (1 31% 5’-ATCCCACTACGTCGATACA-3' ‘USEF 31 W 5'-TGGAAGTAAGTGAGCGTIAT-3 3 7 PCR TH, BA: 94C BR HE S min; 94 Bt 30 9,55°CSEtE 30 5,72°C HEH 30 5, BEI 35 RIF; 4RIE 72°C HRI 10 min 1.2.2.4 Southern 3 3€ 4 OF 20 pe BFE ME DNA, Hind HEA MME T 37 CRT R. RCAF SERED EFT BW BT Fy 0 BK SERRE HEIR. BRET FBN 3&1 500 bp HEL, FA WG #13 MRC BIB ( Primer-a-Gene Labeling System, Promega) 3 a-"P-dCTP #RICHRET » FRICHERY El Sigma 437), RG OT, MM 2~ 4h, RARE, AD 2x SSCHI 0.05% SDS BE A Mi BEAR 2 ~ 3 tk, FE 40 min, #85 FH 0.2 x SSC #1 0.1% SDS BE SOT HERE 2- 3K, FE 40 ming BEAR RAVE RR SE CLM EAE BE, HE Xray BEF - 70C EAT A HAGW 2d Re EUMe. 1.2.2.5 Northem 4305} 6 SE Hi ATE RNA BSE KMLI4A). 15 pg 9 ~ 12 d HYSTA RNA AF Northem 3e204}4F 0 BCS} He he)" HAT RNA BH ERE FM. CCRRIDUL. Met Ric, RL Fe TEAR AE Hr B97 HEF] Southem 2&2E 1.2.3 $A ATE SE Se MH a BRA FREE A T, FRE Ty FRAY FEAR PR A a BRS FR BE Ab BY Be A HE ESE BE AR LB A HE A A Po AT HF 9 hE HIVI900( ICC Pree) 2 BRED 2.1 T REBRRSRHRBRSH CEARLA ETE -MSNHER HAA ERNE iN HE eB HE ST 699, BF OHA RES HAROEERHED ES BES4ITHE BAMA OTA. RAMEE READ) 3 ~ 4 HA BS RRR AE PRE — POR WT. MEER RAR RDU UETT T 2 HES AL BESS , RB Be SAAT 500 SR AE BGR A 50 TAR ACF He PE HR 80 Ri. HFM MART EAT, RE PERL 20% 24 A FST LT ETT SR HHL LLL a A AGF 9 20 BRT, ACE PRET T BB RM CUS ML AL PCR KM APT. HOP EB A RA 17 PRIA HE, CUS. 82M 13 RIB TE, B02 PBN PCR #23 14 REE TE TEMS 70%. HPAL, mpt HI, CUS HE AI A RT RA — HE KE HAART TERI, TALIM AY 20 HRT, AOE SERRE 3 ARE IU Hk a HEM 13 RC 1), 2 BAR TTA te FBN MEA PCR HAR SWERRAC SRA RMME RIS H. AL 1, ROMRRRRO ‘Table 1 Analysis of T, transgene plants CUS AEE ‘GUS histochemical staining mo ano TERRE No. Line PCR Hitt ayee PR are T 2 3 ‘ 5 6 1 3 9 10 u 2 B rm 1s 16 7 18 rr 20 + spostve plant; — cneguve pla 2.2, T, #4835 HA 749 Southern blot #1 Northern blot #8) AMER 4 PT, RARE A RE MAAS DNA, FA Hind ll MEW, UA PBN AEB S'tii 500 bp HELPER Et ZE4T Southem JR. HRA BAC ENS), Sh UR PBN 3 AE Me OE AR EE