Nghiên cứu phát triển hệ thống chuyển gen bằng vi khuẩn Agrobacterium tumefaciens cho nấm sợi Aspergillus oryzae

I H C QU C GIA HÀ N I TR NGă IăH CăKHOAăH CăT ăNHIểN KHOAăSINHăH C Nguy năTh ăKhuy n NGHIểNăC UăPHÁTăTRI NăH ăTH NGăCHUY Nă GENăB NGăVIăKHU NăAgrobacterium tumefaciens CHOăN MăS IăAspergillus oryzae Chuyên ngành: Vi sinh v t h c Mã s : 60420107 LU NăV NăTH CăS ăKHOAăH C Ng iăh ngăd năkhoaăh c:ăTS.ăTr năV năTu n HƠăN iă- 2016 M CL C Trang M Ch Đ U ng T NG QUAN 1.1 Gi i thi u chung v n m s i Aspergillus oryzae Đ c m sinh h c c a n m s i Aspergillus oryzae Đ c m di truy n c a n m s i A oryzae Vai trò c a n m s i A oryzae Ph}n bi t Aspergillus oryzae v i Aspergillus flavus sinh đ c t aflatoxin C|c ph ng ph|p chuy n gen v{o n m s i 12 Ph ng ph|p chuy n gen s d ng t b{o tr n (protoplast) 12 Ph ng ph|p chuy n gen thông qua vi khu n A tumefaciens (ATMT) 13 1.2.2 K thu t chuy n gen b ng xung n (electroporation) 13 1.2.4 Marker s d ng ch n l c c|c ch ng chuy n gen 17 1.2.5 M t s gen ch th dùng chuy n gen Ch ng V T LI U VÀ PH vi n m 19 NG PHÁP 23 Nguyên li u 23 2.1.1 Ch ng vi sinh v t 23 2.1.2 Thi t b v{ hóa ch t 25 Môi tr Ph ng s d ng nghiên c u 28 ng ph|p nghiên c u 28 2.2.1 Thu nh n b{o t n m 29 T|ch chi t DNA RNA v{ t ng h p cDNA 29 Quan s|t hình th|i v{ ki m tra m t s đ c m sinh lý sinh hóa 30 Ph}n bi t A oryzae v{ A Flavus b ng c|c k thu t sinh h c ph}n t 31 2.2.5 T o c|c vector nh th dùng cho chuy n gen 32 2.2.6 Chuy n gen v{o n m s i A oryzae Error! Bookmark not defined Ch ng K T QU VÀ TH O LU N Error! Bookmark not defined X|c nh n ch ng A oryzae an to{n đ s d ng cho chuy n gen Error! Bookmark not defined Ph}n bi t c|c ch ng A oryzae an to{n v{ ch ng Aspergillus flavus sinh đ c t aflatoxin Error! Bookmark not defined 3.1.2 L a ch n ch ng A oryzae ph c v cho chuy n gen Error! Bookmark not defined 3.2 L a ch n marker chuy n gen v{o A oryzae Error! Bookmark not defined X}y d ng h th ng chuy n gen thông qua vi khu n A tumefaciens Error! Bookmark not defined Xóa gen pyrG đ t o ch ng đ t bi n tr d ng uridine/uracil Error! Bookmark not defined 3.3.2 T o m t s vector nh th dùng cho chuy n gen v{o A oryzae tr d ng uridine/uracil Error! Bookmark not defined Đ|nh gi| hi u qu c a h th ng chuy n gen v i gen huỳnh quang GFP v{ DsRed Error! Bookmark not defined 3.4.1 Chuy n gen GFP v{ DsRed v{o n m s i A oryzae tr d ng uridine/uracil Error! Bookmark not defined X|c nh n c|c th chuy n gen Error! Bookmark not defined 3.5 ng d ng h th ng chuy n gen m i thi t l p đ bi u hi n gen phyA m~ hóa enzyme phytase c a A fumigatus A oryzae Error! Bookmark not defined K T LU N VÀ KI N NGH Error! Bookmark not defined TÀI LI U THAM KH O 34 M U Aspergillus oryzaelà loài n m s i đ ph m truy n th ng đ u ng t i nhi u n c s d ng r ng rãi s n xu t th c c châu Á bao g m Nh t B n, Trung Qu c, Hàn Qu c, Vi t Nam, Thái Lan, Malaysia Philippines Loài n m đ c C c Qu n lý Th c ph m D (GRAS) Vi t Nam, A oryzae đ s đ a ph ng c ph m Hoa K (FDA) công nh n an toàn c s d ng đ s n xu tt ng truy n th ng t i m t t nh phía B c.A oryzae có kh n ng ti t m t l enzyme khác vào môi tr t bào đ s n xu t th ng Do đó, loài n m đ ng l n c s d ng nh nhà máy ng m i nhi u lo i enzyme protein c d ng t nhiên tái t h p G n ch ng A oryzae RIB40 dùng s n xu t công nghi p t i Nh t B n đ c gi i trình t toàn b h gen S hi u bi t t ng t n v h gen c a A oryzae m nhi u c h i c i bi n di truy n bi u hi n gen loài n m s i an toàn Hi n nay, hai ph ng pháp ph bi n nh t chuy n gen vào n m s i chuy n gen qua t bào tr n chuy n gen thông qua vi khu n Agrobacterium tumefaciens Tuy nhiên vi cchuy n gen vào n m s iA oryzaecho đ n ch s d ng ph ng pháp chuy n gen qua t bào tr n.Ph ng pháp t ng đ i ph c t p t n kém, khó có th th c hi n t i u ki n phòng thí nghi m t i Vi t Nam Ph ng pháp chuy n gen s d ng vi khu n A tumefaciens đ n gi n h n v i chi phí h p lý Tuy nhiên ch a có m t nghiên c u báo cáo v vi c chuy n gen thành công n m s i A oryzae Bên c nh đó, A oryzaecókh n ng kháng l i h u h t h p ch t kháng sinh th ch ng A oryzae tr d ng đ c s d ng cho chuy n gen.Do phát tri n ngđ s d ng cho chuy n gen gi i pháp nh t đ c i bi n di truy n c ng nh bi u hi n gen A oryzae V i m c đích xây d ng h th ng chuy n genhi u qu cho n m s i A oryzae thông quavi khu n A tumefaciensnh m ph c v h ng nghiên c u b n v ng v bi u hi n enzyme/protein tái t h p loài n m này, th c hi n đ tài“Nghiênă c uă phátătri năh ă th ngăchuy nă gen b ngă viă khu nă Agrobacterium tumefaciens choăn măs iăAspergillus oryzae” v i nh ng n i dung nh sau: - Xác nh n ch ng A oryzae an toàn, có th s d ng làm đ i t ng chuy n gen - T o ch ng A oryzae đ t bi n tr d ng uridine/uracil b ng cách xóa gen pyrG - T om t s vector nh th (binary vector) s d ng cho chuy n gen bi u hi n gen n m s i A oryzae ánh giá hi u qu c a h th ng chuy n gen s d ng gen ch th hu nh - quangGFP vàDsRed - B c đ u ng d ng h th ng chuy n gen m i t ođ c đ bi u hi n gen phyA mã hóa enzyme phytase t n m s i Aspergillus fumigatus Ch ngă1.ăT NG QUAN 1.1 Gi i thi u chung v n m s iAspergillus oryzae 1.1.1 căđi măsinhăh căc aăn măs iAspergillus oryzae N m s iA oryzae thu c phân nhóm Flavic achi Aspergillus, h Trichocomaceae A oryzae có c u t o đa bào, đ c nuôi c y môi tr ng đ a th ch,h s icó màu tr ng xámsau phát tri n chuy n sang màu vàng nh t đ n xanh H s i n m A oryzae sinh tr th ng r t nhanh phân m nh, chi u ngang có kích c 5-7 µm Trên nh ng s i hình thành c u trúc sinh s n vô tính, g i cu ng sinh bào t Cu ng sinh bào t c a A oryzae th ng dài 1-2 mm Trên cu ng ph ng lên t o thành b ng, t b ng m c lên t bào nh , thuôn dài hình chai, x p sát nhau, g i th bình; th bình có đính nh ng chu i bào t màu vàng l c hay màu vàng hoa cau (còn g i bào t đính)[31, 53, 80, 99] Hình thái khu n l c cu ng sinh bào t c a n m s i A oryzae đ Hình 1.1 c Machida c ng s mô t chi ti t nh H̀nhă1.1.Hình thái khu n l c c u trúc cu ng sinh bào t c a n m s i Aspergillus oryzae RIB40[53] N m s iA oryzae sinh tr tr ng t t nhi t đ 30ºC-40ºC, chúng có th sinh ng phát tri n d dàng nhi u lo i c ch t khác nh m nh v n th c v t, lá, g m c nát, lo i h t, th c n gia súc A oryzae phân b r ng rãi toàn th gi i, đ c bi t ph bi n n phát tri n, A oryzae ti t môi tr c nhi t đ i Trong trình sinh tr ng m t l ng ng l n enzyme th y phân nh cellulase, pectinase, xylanase, amylase, protease, glucoamylase[36, 53] 1.1.2 căđi mădiătruy năc aăn măs iăA oryzae G n đây, ch ng A oryzaeRIB40 đ c s d ngtrong công nghi p s n xu t th c ph m lên men truy n th ng t i Nh t B n đ c gi i trình t toàn b h gen, theo h genc a A oryzaeg m nhi m s c th v i t ng kích th c 37 megabase (Mb), d đoán có 12.074 gen mã hóa protein, l n h n 25%-30% so v i loài khác thu c chi Aspergillus Ch ng h n,h gen c a A oryzae l n h n loài A nidulans(loài chu n dùng cho nghiên c u phòng thí nghi m) A fumigatus(tác nhân gây b nh c h i ng i) l n l t 29% 34%[53].H genc a A oryzae l n h nch y u thu n p thêm DNA trình ti n hóa Vi c m r ng kích th d c h gen ng nh đ c tr ng cho loài có quan h g n g i v i A oryzaenh A niger A flavus, loài cókích th c h gen t r t g n g i v i A oryzae A flavuscó đ t ng đ ng c bi t loài có quan h ng đ ng DNA đ n 99,5%[2, 53, 85] A oryzae có kh n ng phân gi i m nh nhi u lo i c ch t có t nhiêndo loài s h u gen mã hóa enzyme th y phân khác Các loàiA oryzae, A fumigatusvà A nidulans ch a l n l t 135, 99 90 gen mã hóa cho protease, chi m kho ng 1% t ng s gen h gen T t c gen mã hóa protease đ c tìm th y A fumigatus và/ho cA nidulansđ u có m t A oryzae, nhiêncác gen mã hóa m t s enzyme aminopeptidase có m t trongA oryzael i m t h gen c aA fumigatus A nidulans Ngoài ra,A oryzaecòn s h u thêm nhi u gen mã hóa protease ti t có kh n ng ho t đ ng môi tr ng có tính axit S gia t ng protease ho t đ ng pH axit c aA oryzaecó th hình thành trình thu n hóa c a ng i [53].S hi u bi t t ng t n v h gen c a Aspergillus oryzae m nhi u c h i m i nghiên c u c i bi n di truy n ng d ng loài n m an toàn s n xu t s n ph m nh m ph c v m c đích ng i 1.1.3 Vaiătròăc aăn măs iA.oryzae N m s iA oryzaeđ c thu n hóa t nh t 2000 n m tr c hi n đ s d ng r ng rãitrong s n xu t th c ph m Nh t B n, Trung Qu c n Á nh lên men đ u nành, làm n c s t, t phânnh tr c ông ng m t s đ u ng có c n nh huangjiu, sake, makgeolli shochu[4, 36, 111] A oryzae đ th c ph m đ c tính sinh tr c ng nhanh,ti t l c s d ng r ng rãi ng l n enzyme th y amylase, glucoamylase, carboxypeptidase, protease trình sinh ng t o mùi th m d ch u cho s n ph m lên men[37].Trong lên men truy n th ng, A oryzae th ng đ c nuôi môi tr phát tri n hi u khí c a s i n m ng r n, u ki n thích h p cho s m thích h p trình lên men môi tr ng r n giúp t ng kh n ng s n sinh enzyme ch t chuy n hóa mà th ng s không đ c s n xu t u ki n lên men l ng[99] Ngoài vai trò s n xu t th c ph m truy n th ng, n m s i A oryzae đ c s d ng m t s ngành công nghi p s n xu t enzyme có giá tr kinh t cao nh glucoamylase, -amylase m t s protease ph c v s n xu t bánh k o, đ u ng Glucoamylase -amylase thu c nhóm enzyme amylase, chi m t i g n 20% t ng s n l ng enzyme đ c s n xu t, đóch y u đ c s n xu t b in m s iA oryzae[16, 81, 98, 113].Ngu n cung c p enzyme protease cho công nghi p s n xu t th c ph m ch t t y r a đ n t loài n m s i thu c chi Conidiobolus, Verticillium, Penicillium Aspergillus, ph i k đ n loài sinh protease r t cao A oryzae[1, 106] Bên c nh nh ng l i ích v kinh t , A oryzae đ c coi vi sinh v t mô hình dùng cho nghiên c u v bi u hi n enzyme protein tái t h p Ngoài nghiên c u chuy n gen h tr vi ctìm ch c n ng c ng nh vai trò c a gen m i[54] 1.1.4 Phơnăbi tăAspergillus oryzae v iAspergillus flavussinhăđ căt ă aflatoxin Hi n nay, t l ng i m c b nh ung th t ng lên ngày m t t ng cao M t nh ng nguyên nhân n ph i th c ph m ch a ch t gây ung th , cóđ c t n m aflatoxin c t aflatoxin đ c sinh b i n mm cAspergillus flavusvà Aspergillus parasiticustrên lo i h t nông s n quan tr ng không đ qu n t t nh g o,l c, ngô, đ u t loàiA oryzaemà th ng đ cb o ng Hình thái c aA flavusg n nh gi ng h tv i c s d ng ngành công nghi p th c ph m[17, 26, 80] A flavusvà A oryzaelà hai loài thu c phân nhóm Flavi c a chi Aspergillus T ng t nh A oryzae, n m s i A flavus có kh n ng t n t i 48ºC sinh tr flavus t n t i oryzae, d ng t i u nh t nhi t đ t 12ºC đ n 28ºC đ n 37ºC H u h t chu trình s ng A d ng h s i sinh s n b ng bào t đính.Gi ng v i m t s ch ng A i u ki n b t l i, s i n m A flavus chuy n thành c u trúc đ c bi t g i h ch n m (sclerotia) H ch n m giúp chúng có th t n t i d i u ki n kh c nghi t Khi u ki n tr nên thu n l i, h ch n m s phát tri n thành d ng s i vàt sinh cu ng mang bào t đính, phát tán môi tr ng đ t không khí [17, 26] Trong t nhiên, r t khó đ có th phân bi t hai loài A oryzae A flavus b i hình thái r t gi ng nhau.H n n a,khi gi i trình t hai ch ng đ i di n cho hai loài, nh n th y genome c a ch ng A oryzae RIB40 A flavus NRRL 3357 có đ t ng đ ng 99,5% DNA 98% protein [85] Phân tích ti n hóa m t s ch ng thu c hai chi này, nh n th yA flavus A oryzaecó m c đ t ng đ ng tùy thu c vào t ng ch ng Ví d nh hai ch ngA flavusSRRC 1357 SRRC 2112có m i quan h g n g i v i A oryzae h n ch ngA flavus khác, có nhi u đ c m c a A oryzae h n (Hình 1.2) T d li u v h gen, A oryzaeđ c cho r ng có ngu n g c phát sinh t loài A flavus[18] H̀nhă1.2.M i quan h phát sinh loài s khác bi t h gen c aA oryzae A flavus (màu xanh lam A oryzae, màu xanh A flavus)[18] A oryzaekhác v i A flavus trình sinh tr m t đo n vi c m t kh n ng sinh đ c t aflatoxin ngdo trình ti n hóa tích l y nhi u đ t bi n m đ t bi n m t s gen liên quan đ n sinh t ng h p aflatoxin[8] Con đ ng sinh t ng h p aflatoxin b t ngu n t axit norsolorinic (NOR), tr i qua nh t 23 trình chuy n hóa trung gian, nh enzyme đ c mã hóa b 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tumefaciens 1.2.3.1 C ch chuy n gen vào t bào ch c a A tumefaciens Vi khu n A tumefacienslà m t loài vi khu