Phân lập và thiết kế vector mang promoter liên quan đến quá trình phát triển phôi ở cây arabidopsis

I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN Nguyn Hựng Mnh PHN LP V THIT K VECTOR MANG PROMOTER LIấN QUAN N QU TRèNH PHT TRIN PHễI CY ARABIDOPSIS LUN VN THC S KHOA HC H Ni - 2015 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN Nguyn Hựng Mnh PHN LP V THIT K VECTOR MANG PROMOTER LIấN QUAN N QU TRèNH PHT TRIN PHễI CY ARABIDOPSIS Chuyờn ngnh: Sinh hc thc nghim Mó s: 60420114 LUN VN THC S KHOA HC NGI HNG DN KHOA HC: TS Lờ Hng ip H Ni - 2015 LI CM N hon thnh lun ny, li u tiờn tụi xin by t lũng bit n chõn thnh v sõu sc ti TS Lờ Hng ip ó tn tỡnh hng dn v dỡu dt tụi sut quỏ trỡnh nghiờn cu v thc hin lun Tụi xin chõn thnh cm n lónh o trng i hc Khoa hc T nhiờn i hc Quc gia H Ni v cỏc thy cụ giỏo trng ó ging dy v to iu kin cho tụi hc v thc hin lun Tụi xin by t lũng bit n n cỏc thy cụ giỏo phũng thớ nghim Cụng ngh nuụi cy mụ t bo thc vt, cỏc thy cụ giỏo v cỏc anh ch b mụn Sinh lý thc vt v Húa sinh, cỏc thy cụ giỏo khoa Sinh hc, trng i hc Khoa hc T nhiờn - i hc Quc gia H Ni ó truyn dy kin thc v k nng cho tụi sut quỏ trỡnh hc di mỏi trng Khoa hc T nhiờn, to iu kin thun li tụi cú th thc hin tt ti nghiờn cu ca mỡnh Cui cựng, tụi xin gi li cm n ti bn bố, ng nghip cựng th lp Sinh hc thc nghim khúa 2012 - 2014, nhúm sinh viờn phũng thớ nghim, nhng ngi ó luụn sỏt cỏnh bờn tụi, giỳp , ng viờn v gúp ý cho tụi sut quỏ trỡnh hc v hon thnh ti ny Tụi xin chõn thnh cm n! H Ni, ngy 24 thỏng 11 nm 2015 Hc viờn Nguyn Hựng Mnh M U Ngy nay, khoa hc k thut phỏt trin vt bc gn lin vi i sng ngi, hin din mi lnh vc vi mc ớch nõng cao cht lng cuc sng Cụng ngh sinh hc i ỏnh du bc tin mi nht n lc lõu di chinh phc t nhiờn ca ngi, c bit l s phỏt trin ca cụng ngh sinh hc hin i nhiu nm gn õy S dng cụng ngh ADN tỏi t hp, cỏc nh khoa hc ang c gng to nhng cõy trng vt nuụi cú nng sut v cht lng cao, nhng thc phm, dc phm phc v cha bnh cho ngi Hin nay, cụng ngh t bo v k thut chuyn gen ang rt phỏt trin Vit Nam Vit Nam l mt nc nụng nghip truyn thng, vỡ vy vic phỏt trin v ng dng cụng ngh sinh hc c trung ch yu vo nụng - lõm - ng nghip, vi mc ớch a nn nụng nghip nc ta tr thnh nn nụng nghip hin i, ng dng khoa hc k thut vo sn xut tng cht lng, nng sut sn phm, ng thi gim thiu sc lao ng ca ngi Vit Nam nm vựng khớ hu nhit i núng m, ma nhiu, khỏ thun li nhiu loi ng, thc vt tn ti v phỏt trin Theo cỏc nh khoa hc thỡ Vit Nam l quc gia ng th 16 trờn th gii v a dng sinh hc, riờng ngnh thc vt bc cao ó cú khong 12000 loi thuc hn 2500 chi v 300 h [12] Trong ú cú rt nhiu loi cú giỏ tr, ỏp ng c cỏc nhu cu khỏc ca ngi nh lng thc thc phm, lm thuc, cõy cnh, Trong cụng ngh chuyn gen thc vt, vic thit k vector biu hin l mt khõu quan trng v úng gúp rt ln cho s thnh cụng ca cụng ngh ny Vỡ vy vic tỡm kim, phõn lp cỏc promoter v cỏc gen cú giỏ tr cú ý ngha vụ cựng quan trng cụng tỏc nghiờn cu v chn lc ging t c hiu qu cao cụng tỏc ny ngi ta thng nghiờn cu trờn nhng sinh vt mụ hỡnh, ú cú Arabidopsis Arabidopsis (Arabidopsis thaliana L.) mc dự khụng cú ý ngha gỡ v mt nụng nghip, nhng nghiờn cu nú úng mt vai trũ rt quan trng Arabidopsis ó c chn nh l mt cõy mụ hỡnh nghiờn cu thc vt v ó c cỏc nh khoa hc trờn th gii s dng t lõu Arabidopsis cú b gen nh ch khong 125 Mb, ó hon ton c gii trỡnh t v cụng b nm 2000 Chu k sinh trng ca Arabidopsis ngn (khong 6-8 tun), cho nhiu ht v d dng nuụi trng mt khụng gian hp v rt hiu qu vic s dng phng phỏp chuyn gen bng vi khun Agrobacterium S hỡnh thnh v phỏt trin ca ht nhiu yu t quy nh ú cú cỏc hormone thc vt nh ABA, GA nhng hormone ny u tham gia hot húa nhiu gen liờn quan n s phỏt trin ca ht T nhng lý trờn, chỳng tụi tin hnh thc hin ti: Phõn lp v thit k vecter mang promoter liờn quan n quỏ trỡnh phỏt trin phụi cõy Arabidopsis, vi cỏc mc tiờu c th nh sau: Thit lp c ngun mu cõy Arabidopsis in vitro Nhõn dũng c trỡnh t promoter liờn quan n quỏ trỡnh phỏt trin phụi ht ca cõy Arabidopsis ó thit k c vector tỏi t hp pBI121-ET cú mang trỡnh t promoter ET v tin hnh a vo cõy Arabidopsis ging Columbia Bc u phõn tớch trỡnh t ca promoter ET Chng TNG QUAN 1.1 Gii thiu v cõy Arabidopsis 1.1.1 Ngun gc v v trớ phõn loi Arabidopsis (tờn Latin Arabidopsis thaliana) l mt loi thc vt cú hoa nh thuc h Ci (Brassicaceae), b Brassicales [38] Arabidopsis cú ngun gc t Chõu u, Chõu v Tõy Bc Chõu Phi Arabidopsis ln u tiờn c phỏt hin nm 1577 dóy nỳi Harz bi mt bỏc s ngi c Johannes Thal (1542-1583) v ó c gi l Pilosella siliquosa n nm 1841, danh phỏp ó c i thnh Arabidopsis thaliana bi mt nh thc vt hc ngi c Gustav Heynhold vinh danh Thal Cỏc loi Arabidopsis cú th c tỡm thy t nhiờn hu nh khp mi ni Bc bỏn cu Arabidopsis ó c tỡm thy mi cao t mc nc bin cho n dóy nỳi cao Himalayas, t phớa bc Scandinavia n Bc Phi, bao gm c Cape Verde Islands ti v 16 Nú cng c tỡm thy Bc M, cú th l bt ngun t Chõu u [38] 1.1.2 Sinh trng v phỏt trin Arabidopsis cú chu kỡ sng tng i ngn so sỏnh vi cỏc loi thc vt khỏc Ton b chu trỡnh sng bao gm s ny mm ca ht, to thnh cõy hỡnh hoa th, mc thõn chớnh, hoa v thi sinh trng n hỡnh thnh ht l khong n tun Arabidopsis cú kớch thc rt nh, cõy phỏt trin thnh cm hỡnh hoa th ng kớnh 2-10 cm, hoa di mm, ht trng thnh t kớch thc 0.5 mm v chiu di Lỏ c bao ph bi lụng n bo nh gi l trichomes thun tin cho nghiờn cu hỡnh thỏi v s khỏc bit ca t bo Lỏ tht th nht cú trichomes ch b mt trờn ca lỏ, cỏc lỏ cũn li thỡ cú c mt di S lng lỏ hỡnh hoa th c hỡnh thnh ph thuc vo kiu gen v iu kin mụi trng cựng vi s tng quan mnh vi thi gian t lỳc ny mm n lỳc bt u hoa [38] Ht ging Arabidopsis nh, cú hỡnh bu dc Ht ging cú th ny mm mt cỏch d dng, mc dự ht sau thu hoch thỡ cn c x lý lnh mt vi ngy v lu tr bo qun mt thi gian ht cú sc ny mm tt nht Ht thng yờu cu ỏnh sỏng ny mm Ht cõy Arabidopsis c gieo trờn a petri hoc chu t nh kớnh hoc di ỏnh ốn hunh quang phũng thớ nghim Khong tun sau gieo ht, thõn chớnh s bt u phỏt trin, to cm hoa v sau ú l qu Hoa cu to phớa ngoi l bn i hoa, vũng xon bờn cú bn cỏnh hoa mu trng, sỏu nh hoa mang phn hoa v mt nhy trung tõm cú hai lỏ noón hỡnh thnh nờn qu Arabidopsis l loi thc vt t th phn, cõy trng thnh t chiu cao 15-20 cm R cú cu trỳc n gin, khụng cng sinh vi cỏc vi khun c nh m, to iu kin d dng cho vic nghiờn cu mụi trng ti u cho sinh trng [38,47] 1.1.3 c im v di truyn Arabidopsis l loi thc vt u tiờn, v l sinh vt a bo th ba sau Caenorhabditis elegans v Drosophila melanogaster hon ton c gii trỡnh t gen [15,55,56] H gen ca Arabidopsis cú kớch thc 125 Mb mó húa cho khong 26000 gen trờn nhim sc th [2] Theo mt bỏo cỏo khỏc ca Hall v cng s [31] h gen ca Arabidopsis cú kớch thc khong 146 Mb mó húa cho khong 25000 gen K t h thng gii trỡnh t gen Arabidopsis c hon thnh v cụng b nm 2000 thỡ trỡnh t cỏc gen ngy cng c nghiờn cu m rng, c b sung v hon thin Cỏc vựng gen ca h gen ó c gii trỡnh t v phõn tớch khong 119 Mb Trỡnh t h gen c phõn tớch bi TIGR (The Institute for Genome Research) v5 analysis (http:// www.tigr Org/tdb/e2k1/ath1/ath1.shtml) [19,30] Arabidopsis l loi thc vt cú b gen nh nht, vi ớt trỡnh t lp li nht so vi bt k loi thc vt bc cao no c bit n, to iu kin cho rt nhiu nghiờn cu phõn t da trờn bn tỏch dũng Ngoi ra, Arabidopsis cú th d dng c chuyn gen nh vi khun Agrobacterium tumefaciens, ú l iu kin tiờn quyt cho nhiu thớ nghim di truyn phõn t [51] Arabidopsis c xut lm thc vt mụ hỡnh ln u tiờn bi Friedrich Laibach nm 1943 Mc dự thc t Arabidopsis cú rt nhiu c im thun li cho phõn tớch di truyn hc nhng ó khụng lp tc c s dng ph bin 1.1.4 Giỏ tr ca cõy Arabidopsis Arabidopsis l thc vt mụ hỡnh rt hu ớch cho nghiờn cu di truyn hc v sinh hc phõn t thc vt, bao gm nghiờn cu bnh lý hc v sinh lý hc, vi nhiu hiu bit t ú cú th ỏp dng trc tip cho cõy trng Trong tt c cỏc loi thc vt cú hoa, cõy Arabidopsis c s dng nhiu nht lm i tng cho cỏc nghiờn cu, hn hn tt c cỏc loi khỏc tng c coi l ng c viờn y hn nh hng nghiờn cu thc vt tng lai Ch riờng nm 2008, ó cú hn 3500 ti liu cú liờn quan n Arabidopsis c thờm vo c s d liu Pubmed So vi nm 1979, ch cú cụng b v Arabidopsis v 65 cụng b cho tt c cỏc nm trc cng li S phỏt trin ca vic nghiờn cu Arabidopsis vũng 30 nm qua rt ỏng chỳ ý, b ớch, cú tỏc dng to nờn mt bc tin mi cụng ngh sinh hc thc vt [37] Vic nõng cao vai trũ ca di truyn hc thi k hi nhp cựng vi s sn cú ca cỏc cụng c sinh hc phõn t ó dn n vic cỏc nh thc vt hc trung vo mt sinh vt phõn tớch chi tit Arabidopsis mó húa cho cỏc gen cựng ngun gc ca hu ht cỏc protein c cỏc sinh vt nhõn tht s dng trỡ tớnh ton ca b gen [55] S la chn nghiờn cu Arabidopsis ó mang li v hn s mang n nhng hiu bit mi thỳ v Arabidopsis cú mt s lng ỏng k cỏc t bin c trng, nhiờn vic nghiờn cu tỡm kim cỏc t bin mi cng rt quan trng Ht ging Arabidopsis rt nh l tng i nhy cm vi cỏc cht gõy t bin húa hc, ú, vic tỡm kim cỏc t bin thng xuyờn c bt u bng cỏch gieo hng ngn ht ging x lý vi ethyl methane sulfonate (EMS) i vi nhiu mc ớch khỏc, ht ging n gin l cú th trng trờn t, 16 ht vi mi chu cm, hoc 250 vi mi mt phng 25 cm x 50 cm, lm gim nhu cu v phng tin vụ trựng la chn ging khỏng thuc hoc thuc dit c, hoc to thun li cho s hp thu cỏc hp cht húa sinh, ht ging c trng trờn mụi trng thch vụ trựng a petri hoc hp nha cho phộp s phỏt trin nhiu hn Lỏ mm xut hin sau ngy, v lỏ tht u tiờn sau ngy Ti thi im ny, cõy ging khỏng thuc cao 5mm, mc lờn t khong 2000 ht ging gieo trờn mi a 10 cm, cú th c xỏc nh v chuyn vo t, hoc tip tc x lý vi ỏnh sỏng UV hoc cỏc tỏc nhõn gõy c khỏc Tip tc phỏt trin thờm nhiu lỏ hn, phỏt trin lỏ hin cú, ADN lỏ s tng tuyn tớnh cho n khong 20-25 ngy sau ny mm [24], v lỳc ú mi cõy s nhanh chúng phỏt trin mt thõn chớnh, chiu cao lờn n 20cm, m hoa v cui cựng l v ht s xut hin vo khong 28 ngy S t th phn l t ng, nhng cng cú th d dng thc hin nh tỏc nhõn bờn ngoi bng cỏch chuyn phn hoa cho nhy hoa ó c loi b bao phn Thụng thng, vic lai cn huyt t ng lp i lp li s lm cho mi dũng Arabidopsis hon ton ng hp t Ch sau tun, cú n 5000 ht ging c thu hoch t mi cõy, v chu kỡ mi cú th c bt u Sn lng ht ging cao lm cho nú cú th dựng nhõn dũng lý thuyt m kh nng sinh sn 99%, hu nh khụng gii hn, ht nh cú th c lu tr cỏc bỡnh hỳt m nhit lnh nu cn thit Cỏc t bin th h u tiờn phỏt sinh t ht M1 hoc t on chốn T-ADN (T1) l gen ln v khụng cú kiu hỡnh Lai Menden cỏc t bin cú th c quan sỏt thy cỏc th h chỏu (M2 hoc T2) Nh vy ch tun sau t bin, mt phn t cỏc th h chỏu M2 phỏt sinh t mt ht ging sn xut t bin mang tớnh cht ng hp t v kiu hỡnh [32] Arabidopsis cú chu kỡ sng ngn, kớch thc cõy nh ú hn ch c mc thp nht cỏc yờu cu v c s vt cht v iu kin sinh trng Ngoi cõy Arabidopsis cũn cú kh nng sn sinh nhiu ht mi th h Vỡ cỏc tớnh nng ny m t ban u 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