p h An t i c h d U l ie u c u a p r o t e i n g i Au m e t h i o n i n e THONG QUA SANG LOC HE PROTEIN CUA SAN Chu Dilc Ha1, Nguyen Ha My2, Nguyen Chi Thanh3, Pham Thi Dung3, Nguyen Quoc Trung3, Pham Phifdng Thu2, Le Thi Ngoc Quynh4, Ha Thi Quyen1, Le Thi Hien1, La Viet Hong2 t m tat Trong nghien ciiu nay, thong tin ve nhom protein giau Methionine (Methionine-rich protein, MRP) da diidc tim hieu mot each day du tren cay san (Manihot esculenta) bang cac cong cu tin sinh hoc Ket qua da xac dinh difoc t6ng so 155 MRP, v6i tieu chi kich thifdc > 95 axit amin va ham 1ifpng Met > 6% Trong do, 52 (tren tong so 155) MRP chifa difdc chu giai chile nang san Phan tich cho thay cac MRP chifa ro chile nang co dac tinh ly hoa da dang Dd doan vi tri phan bo noi bao da chi rang cac MRP co the nam cf luc lap, ty the va he thong bao goi Dang chu y, cac gen ma hoa MRP chifa ro chile nang co bieu hien khac tren cac cd quan chinh tren cay san Ket qua cua nghien ciiu da cung cap nhdng dan lieu quan cho viec tim hieu cd che dap ling bat ldi phi sinh hoc cua cay san Tif khoa: Cay san (Manihot esculenta), protein giau Methionine, dac tinh ly hoa, tin sinh hoc L DAT VAN DE Cac dieu kien bat thuan, nhif bat ldi ve nguon nifdt, bat ldi ve nhiet va nhiem kim loai nang, dridc xem la yeu to gay tac dong 16n den sinh trifcmg l i p h k trien cua cay Cu the, bat ldi phi sinh hoc gay roi loan cac qua trinh sinh ly, dien hinh a h f kim ham kha nang mam, giam quang ho’p, k k n cay cham phat trien va gay thiet hai 50% nang suat Nhom yeu to bat ldi phi sinh hoc m m d aac ghi nhan la nguyen nhan chinh gay nguy hai cho san xuat nong nghiep ben vifng va de doa tinh hinh an ninh ltldng thvfc Viet Nam cap te bao, tac dong cua bat Idi phi sinh hoc lam gia tang qua mile cac dang oxi phan ling (reactive oxygen species, ROS) te bao (Huang et al, 2019) Sd dif thifa cua goc ROS co the tao nhilng bien doi tren phan til cua protein, dac biet la oxi hoa goc Methionine (Met) cau true (Kim et al., 2014) Cac protein giau Met (Methionine-rich protein, MRP) difdc xem la mot nhiing phan tif man SfcooGiogagte Nong nghiep, Dai hoc Cong nghe, Dai hoc Quoc gia Ha Noi PKAanfiah - Kr tiMcat Ndng nghiep, Dai hoc Sif pham Ha Noi agbe Shtfi hoc Hoc vien Nong nghiep Viet Nam;4Bo mon Cong nghe Sinh hoc, Dai hoc Thuy ldi Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/202 cam nhat v6i tac dong cua ROS te bao Tim hieu ve cac MRP cho phep lam ro hdn ve cO che tac dong cua bat ldi phi sinh hoc, tii giai quyet cho bai toan nang cao tinh chong chiu cay trong, nhat la tren cay san (Manihot esculenta), mot nhflng cay quan hang dau fl Viet Nam hien (Malik et al, 2020) Muc tieu cua nghien cflu nham sang loc tat ca cac MRP dfl lieu he protein cua giong san KU50 Thong tin di truyen va chu giai gen sau dupe phan tich dUa tren dfl lieu he gen Dac txnh ca ban va vi tri phan bo noi bao cua cac protein dupe khai thac dfla tren cac cong cu tin sinh hoc Cuoi ding, dfl lieu bieu hien gen ma hoa MRP tai cac ca quan dupe tai phan tich dfla tren thong tin giai trinh tU he phien ma n VAT LIEU VAPHtfONG p h Ap n g h ie n CtfU 2.1 Vat lieu nghien cflu He gen, he protein cua giong san KU 50 (ma dfl lieu: PRJNA234389) tren ca sd dfl lieu Phytozome (Goodstein et al., 2012) va NCBI (Bredeson et al., 2016) 2.2 PhUcfng phap nghien cflu - PhUOng phap tim kiem MRP: Dfl lieu he protein cua san khai thac tren cong NCBI (Bredeson et al., 2016) dflpc sfl dung de sang loc cac MRP bang BioEDIT (Hall, 1999) dUa theo mo ta nghien cflu trUflc day (Chu et al., 2016) Cu the, MRP dfldc dinh nghia la trinh tfl peptide > 95 axit amin (aa) va co ty le Met > 6% (Chu et al., 2016) Toan bo trinh tU dap flng yeu cau dupe khai thac cho phan tich in silico tiep theo - PhUdng phap sang loc va chu giai gen ma hoa protein giau: Trinh tU aa flng vien dfldc truy van (BlastP) vao he protein cua san tren cong Phytozome (Goodstein et al., 2012) va NCBI (Bredeson et al., 2016) nham tim kiem doan gen ma hoa va ma dinh danh tfldng flng Cac trinh tfl aa trung ma dinh danh dupe sang loc thong qua can trinh tU bang ClustalX (Thompson et al., 2002) de lfla chon protein co kich thufle dai hdn (Chu et al., 2016) Vi tri gen tren nhiem sac the dupe xac dinh bang each truy van (BlastN) vao he gen tham chieu tren NCBI (Bredeson etal., 2016) - PhUdng phap phan loai chflc nang cua MRP: Trinh tU aa va ma dinh danh tUdng flng cua MRP dfldc truy van tren MAPMAN (Schwacke et al., 2019) Dfl lieu ve chflc nang cua MRP sau d6 dupe xfl ly tren Microsoft Excel - PhUdng phap phan tich dac tinh cua MRP: Trinh tU aa cua MRP dupe truy van tren Expasy Protparam (Gasteiger et al., 2005) de phan tich dac tinh ve kich thUflc (aa), lUpng phan tfl (kDa), diem dang dien (< 7, axit; > 7, bazd), bat on dinh (< 40, on dinh; > 40, bat on dinh) va fla nUflc trung binh (< 0, Ua nUflc; > 0, ky nUflc) - PhUdng phap dU doan vi tri phan bo noi bao cua MRP: Trinh tU aa day du cua MRP dupe truy van tren cong TargetP and SignalP (Emanuelsson et al., 2007) theo mo ta nghien cflu trUflc day (Chu et al., 2016) Trong do, cac doan tin hieu dac trUng cho bao quan, bao gom he thong bao goi (signal peptide, SP), ty the (mitochondrial transit peptide, mTP) va luc lap (chloroplast transit peptide, cTP) (Emanuelsson et al., 2007) dfloc soat tren dau N-terminal cua MRP - PhUdng phap danh gia bieu hien cua gen ma hoa MRP: Dfl heu RNA-Seq tad 11 mau mo (ma dfl lieu: GSE82279), gom than, choi ben, la, cuong la, gan la, cu, re sdi, mo seo co kha nang phoi hoa (friable embryogenic callus, FEC), to chflc phat sinh phoi soma (somatic organized embryogenic structure, OES), mo phan sinh dinh choi (shoot apical meristem, SAM) va mo phan sinh chop re (root apical meristem, RAM) (Wilson et al., 2017) da dfldc khai thac Theo do, mfle bieu hien cua gen dfldc the hien qua so lfldng trinh tfl doc sfl dung de lap rap nen moi transcript he phien ma tho theo diem so FPKM (Fragments Per Kilobase of exon per Million fragments mapped) Nhflng transcript co diem so FPKM < , dflfli ngfldng phat hien; 10 < FPKM < 30, co bieu hien; 30 < FPKM < 50, bieu hien yeu; 50 < FPKM < 70, co xu huflng bieu hien manh; 70 < FPKM < 100, bieu hien manh; FPKM > 100, bieu hien dac thu (Wilson et al., 2017) 2.3 Thcfi gian va dia diem nghien cflu Nghien cflu dfldc thfle hien tfl thang 11/2020 den thang 02/2021 Cac phan tich in silico dfldc tien hanh tai Hoc vien Nong nghiep Viet Nam, Dai hoc Cong nghe (Dai hoc Quoc gia Ha Noi), Dai hoc Thuy ldi va Dai hoc Sfl pham Ha Noi Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 III KET QUA VA THAO LUAN 3.1 Sang loc va xac dinh protein giau Met he protein cua cay san TrUdc tien, he protein cua san (Bredeson et al, 2016) dUcfc khai thac de sang loc to an bo MRP (kich thUdc > 95 aa va ham lUcfng Met > 6%) (Chu et al., 2016) Ket qua sang loc da xac dinh dUdc tong so 155 phan tP MRP dP lieu cua cay san Trong do, da so cac MRP (149/ 155) dUdc ma hoa bdi gen nhan, MRP dUdc quy dinh bdi gen chvia co th6ng tin chu giai (unplaced scaffold), khong co Dau tirong MRP nao diipc ma hoa bdi gen nam te bao chat Triidc day, 121 va 213 MRP da diipc bao cao tren cay mo hinh la mam Arabidopsis thaliana va dau tiicfng (Glycine max), gen ma hoa MRP nam d te bao chat d Arabidopsis (Chu et al., 2016) Dang chu y, tat ca cac gen ma hoa MRP deu dUdc chu giai thong tin he tham chieu cua hai loai Arabidopsis va dau tUcfng (Chu et al., 2016) Dieu diipc giai thich ban tham chieu cua san chi co dung liipng khoang -582,28 Mb (Bredeson et al., 2016), he gen cua san co kich thiidc thpc te khoang -772 Mb (Awoleye et al., 1994) ArabidoDsis thaliana San Hinh Phan loai chhc nang cua MRP d dau tiicfng, Arabidopsis va san Tiep theo, cac MRP d san diipc phan load chile nang theo tren MAPMAN (Schwacke et al., 2019) Ket qua phan tich cho thay 103/ 155 (chiem 66,45%) gen ma hoa MRP d san da diipc chu giai chile nang, 52/ 155 (chiem 33,55%) gen chiia ro chile nang (Hinh 1) Dang chu y, nghien cilu da chi rang, toan bo cac gen ma hoa MRP chiia biet chile nang d san la nhiing gen dac thu d thpc vat, khong co ket qua tim kiem tiicfng dong (BlastP) doi chieu tren he tham chieu cua cac loai dong vat, tiidng nhii nghien cPu triidc day (Chu et al., 2016) Cu the, 5*/12j (chiem 50,45%) va 92/213 (chiem 43%) gen ma hoa MRP d Arabidopsis va dau tiicfng diipc ghi nhan la chiia co chPc nang (Chu et al., 2016) 3l2 Phan tich dac tinh va vi tri phan bo noi bao caa cac protein giau Met chiia biet chPc nang d san Trong nghien cPu nay, mot so dac diem cd ban ( B MRP chiia biet chPc nang d san da dupe phan tack bang cac cong cu tin sinh hoc Theo do, cac Uriah aa day du cua 52 MRP chUa biet chPc nang d d ajh ao ctru y v in tren Expasy Protparam (Gasteiger cC « L 20QS) de phan tich cac dac diem cau true va J | c fhnh hr hoa cua phan Ket qua khai thac dP 'iin d a o c a i n h boa d hinh Cac MRP chPa biet chPc nang d san co kich thude ngUdng tP 95 (XP_021606075.1) den 279 aa (XP_021628969.1), mPc trung binh dat 148,13 aa (Hinh 2A) Ben canh do, lPpng cua 52 MRP ty le thuan vdi kich thpdc, dao dong tP 10,30 den 32,91 kDa, gia tri trung binh dat 16,84 kDa (Hinh 2A) Cac phan MRP chPa ro chPc nang co gia tri diem dang dien trai dai tP khoang axit (4,43 XP_021628067.1) tdi bazef (10,92 - XP_021629128.1) Trong do, 31/52 (tUcfng Png 59,62%) MRP co pi > (cd tinh bazef) va 21/52 (tUcfng Png 40,38%) phan tP cd gia tri pi < (cd tinh axit) (Hinh 2B) Tiep theo, chi so bat on dinh cua cac protein MRP chPa ro chPc nang nam khoang tP 16,62 (XP_021624467.1) den 90,12 (XP_021633427.1) Nhin chung, da so cac MRP (39/52) bat on dinh 6ng nghiem (do bat on dinh > 40), 13/52 phan tP, vdi chi so bat on dinh < 40, the hien tinh on dinh (Hinh 2C) Gia tri Ua nPdc cua 52 MRP dao dong tP -1,102 (XP_021628075.1) den 1,172 (XP_021623708.1) (Hinh 2D) Phan ldn so (35/52) the hien tinh Pa nUdc (do Pa nPdc dat < 0), 17/52 phan tP co tinh ky nude (do Ua nUdc > 0) Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 A B C D Hinh Dac ti'nh ly hoa, bao gom (A) Kich thddc va ldpng, (B) Diem dang dien, (C) Do bat on dinh va (D) Do da ndPc, cua MRP chda ro chdc nang of san den ty the (cTP), da xac dinh ddpc bon phan tfl (XP_021626513.1, XP_021624445.1, XP_021632296.1 va XP_021606075.1) nam d he thong bao goi (SP) (Hinh 3) Bao cao trdcfc day da chdng minh luc lap va ty the de bi ton thdong tac dong cua sd dd thda ROS (Huang et al., 2019, Kim et al., 2014), nen cac phan td MRP cd tru tai hai bao quan co the la muc tieu de bi de bi oxi hoa dieu kien yeu to bat lpi nhd da ghi nhan nghien cdu trddc day (Chu et al, 2016) 3.3 Phan rich bieu hien cua cac gen m a hoa protein giau Met d cay san cac dieu kien xd ly bat ldi Hinh Dd doan cac trinh td tin hied dac trdng cho vi tri cd tru noi bao cua MRP chda ro chdc nang san Ghi chu: cTP: trinh tU tin hieu co ctkh den luc lap; mTP: trinh tU tin hieu cd dich den ty the, SP: he thong bao goi Tiep theo, cong cu TargetP and SignalP (Emanuelsson et al, 2007) ddpc sti dung de khai thac vi tri cd tru noi bao cua cac MRP chda biet chdc nang d san Theo do, mot phan td MRP (XP_021630288.1) co dich den luc lap (mTP), ba MRP (XP_021602841.1, XP_021611978.1 va XP_021616891.1) co dich Trong nghien cdu nay, dd lieu phien ma tai cac mau mo chinh (Wilson et al., 2017) da ddpc phan tich de tim hieu mdc bieu hien cua cac gen ma hoa MRP chda ro chdc nang d cay san Theo do, mdc bieu hien cua cac gen ma hoa MRP chtla ro chdc nang ddpc bieu hien thong qua bieu nhiet d hinh Ket qua cho thay cac gen ma hoa MRP chiia ro chdc nang co mdc bieu hien da dang d cac cn quan/bo phan chinh cay san Trong do, nghi£n cdu da chi mot so gen co mdc bieu hien manh/ dac thu cac ccf quan/bo phan tren cay n Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 B&J hi$n 95 amino acids and Met > 6% Among them, 52 (out of 155) MRPs have been not annotated and characterized in the proteome of cassava We found that these uncharacterized MRPs exhibited a variation of physic-chemical features Our results also predicted that a number of unknown MRPs could be localized in the chloroplast, mitochrondia and secretory pathway Interestingly, the genes encoding uncharacterized MRP exhibited differential expression in major organs in cassava plants Taken together, our study could provide a critical foundation for further investigation of the mechanism of abiotic stress response in cassava plants Keywords: Cassava (Manihot esculenta), Methionine-rich protein, physic-chemical property, bioinformatics Ngay nhan bai: 20/03/2021 Ngay phan bien: 21/4/2021 Ngiidi phan bien: TS Pham thi Ly Thu Ngay duyet dang: 04/6/2021 ... Nong nghiep Viet Nam - So 05(126)/2021 III KET QUA VA THAO LUAN 3.1 Sang loc va xac dinh protein giau Met he protein cua cay san TrUdc tien, he protein cua san (Bredeson et al, 2016) dUcfc khai... chtla ro chdc nang ddpc bieu hien thong qua bieu nhiet d hinh Ket qua cho thay cac gen ma hoa MRP chiia ro chdc nang co mdc bieu hien da dang d cac cn quan/bo phan chinh cay san Trong do, nghi£n... chila ro chiic nang, co miic bieu hien manh ca ba ccf quan dieu kien thifdng (Chu et al., 2016) Cac ket qua da chi vai tro cua cac MRP lien quan den sinh triidng va phat trien tad mot so vi tri