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Cloning, Mapping and Expression Analysis of β carotene Isomerase Encoding Genes in Wheat (Triticum aestivum L.)

小麦β 胡萝卜素异构酶基因定位克隆及表达分析



全 文 :书!"#$%&
,2016,36(7):1315-1320
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
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CxRTPCRyz{qrβZ[\]^_`|}cDNA(犜犪犇27),~|7€‚ƒ„…†‡ˆ‰jŠ‹ŒA
Ž

‘’
:(1)yz“”2m)•–cDNA—˜,A™š›œ7A、7DžŸj ¡,T¢u犜犪犇277犃犔„
犜犪犇277犇犔;£¤AŽ¥¦,7BžŸ ¡§¨©7ªlm–+«犜犪犇277犅犔。(2)3m犜犪犇27+«¬­®7
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qr
;犇27;žŸš›;+«_;Š‹åæ
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犆犾狅狀犻狀犵,犕犪狆狆犻狀犵犪狀犱犈狓狆狉犲狊狊犻狅狀犃狀犪犾狔狊犻狊狅犳β犮犪狉狅狋犲狀犲犐狊狅犿犲狉犪狊犲
犈狀犮狅犱犻狀犵犌犲狀犲狊犻狀犠犺犲犪狋(犜狉犻狋犻犮狌犿犪犲狊狋犻狏狌犿犔.)
ZHANGPeng1,JIAXiao1,DENGShuai1,SHANLiwei2,FANSanhong1
(1ColegeofLifeScience,NorthwestA&FUniversity,Yangling,Shaanxi712100,China;2ColegeofScience,Northwest
A&FUniversity,Yangling,Shaanxi712100,China)
犃犫狊狋狉犪犮狋:βcaroteneisomerase(D27)isthefirstenzymeinvolvedinstrigolactonesbiosynthesis.Inthis
study,thecDNAsencodingβcaroteneisomerase(犜犪犇27)wereclonedfromwheatcultivarChineseSpring
byRTPCR.Further,theirexpressionindifferenttissuesandunderlowphosphatestresswereanalysed
byRealtimeQuantitativePCR.Theresultshowedthat:(1)Two犜犪犇27cDNAswerecloned,whichwas
mapedonthelongarmsof7Aand7Dchromosome,respectively(named犜犪犇277犃犔and犜犪犇277犇犔).
Inaddition,wefoundanotherlocuslocatedonchromosome7BLencodingthethirdhomolog(犜犪犇27
7犅犔)bysequenceanalysis.(2)Eachofthethree犜犪犇27scontains7exonscomposinganORFof828bp
(7AL),840bp(7BL)or843bp(7DL),respectivelyandencodesaproteincontainingachloroplasttransit
peptideatNterminal.PhylogeneticanalysesindicatedthatD27sinplantsclusteredintothreeindependent
clades.TaD27sshareahighlevelofidentitywithorthologsinothermonocotssuchas犗狉狔狕犪狊犪狋犻狏犪and
犣犲犪犿犪狔狊,asaresult,theyareclusteredinthesameclade.(3)Theexpressionlevelof犜犪犇27isrelative
lyhighinleaves,sheathsandstems,lowerinyoungpanicles,andlowestinroots.Underlowphosphate
stresstheexpressionof犜犪犇27inrootsisdownregulatedtothebottomin6-12hfirstly,andthenitrises
tothenormallevelafter96h.Onthecontrary,theexpressionof犜犪犇27inleavesisupregulatedinearly
stageandthepeakisat6h,thenitcontinuouslydropstoarelativelylowpointat96h.
犓犲狔狑狅狉犱狊:wheat;犇27;chromosomallocalization;genestructure;expressionpattern
  bcdef(SLs)ÏÙ7ç襦,éêu
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IJK*L`7(CCD7)、+Z[\]IJK*L`
8(CCD8)„MNž]P450OPhQ MAX1。2012
ý
,AlderÅ[5]Hš{nCijDHRS},TD27
ôU¼VSæβZ[\]¹¼u9WβZ[\],
XYCCD7„CCD8A™ŒIJ„AB2Z,<
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(carlactone,CL)。2014ýSato
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AtMAX1,n`ôc CL¹¼u+Z[\]efd
(carlactonoicacid,CLA), e f Ò j ¼ g $
(MeCLA)7Ÿ¯ôõSLs5Ÿ³´AtD14fE。
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(犃狋犇27)7 €  ‚ ƒ j Š ‹ å æ õ 犕犃犡3
(CCD7)„犕犃犡4(CCD8)€,7s§(AjŠ
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#¼.;ˆ‰Š&‹
。UmeharaÅ[9]|ÁÂSLs
ghÍû+« 犗狊犇10(CCD8)、犗狊犇17(CCD7)„
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Š‹ÉŒ{.;

¥¦¬¦‰Ñj‘’

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CD

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;ˆ‰Š&‹

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EST•–u—˜,CxS¹™PCR–yz
Öqr犇27(犜犪犇27)+«cDNA,AŽ{+«
_

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A™¥A·Ç

ËÌoN¦§ËÌjÈ

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Ÿ ®¬©j

st

ëB

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st

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ÛjœŒ

\ÜÝÞßàáâãäRNAåæ


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èãäRNAé•。\œyzjcDNAklêjg
hÔ\TaKaRaÕÖjPrimeScriptTMⅡ1stStrand
cDNASynthesisKit,×ØٽڜŒ;\œŠ
‹åæAŽjcDNAjghÔ\ TaKaRaÕÖj
PrimeScriptTM RTreagentKitwithgDNAEraser
(PerfectRealTime),×ØÙ½ÚëÒۜŒ。
1.2.2 45犜犪犇27,=@A o犗狊犇27+«£
¤uG”

7 NCBI§Ô\ Blastìíîïqr
ESTð˜ñ,c”Öj)•–£¤Œò™„•
–

o•–Ò£¤uåóôõ PCR k^ö$
犜犪犇27f(5′AGGCCATAAACATGGAGGCCACCG
CA3′)„犜犪犇27r(5′GCTAGCAATTCACACCAT
AGTCCTGCTTCGCG3′)。oghjcDNAkl
êuåó

Ô\ TaKaRaÕÖj)¬ø` Prime
STARHSDNAPolymerasewithGCBufferŒ
÷ƒ
,PCRø$ùúÒû–ÔpMD19TüŸ,¹¼
ÔSýþ÷ Top10÷j。±Aÿyz!Ô§"
#$<$R%¶&ÕÖ

"

Œä£

1.2.3 犜犪犇27CDEF (\ŸÕš›šÉRT
PCRjœ);犜犪犇277€‚ƒN€Õ*…
†‡ˆ€‰jŠ‹åæ

Θ犜犪犇27ä£Òj
£¤

Ô\BeaconDesigner8ôõ RTPCRö$
犜犪犇27qf(5′GGCTACCAAGGATTAATAGA3′)„
犜犪犇27qr (5′ATCATCACCTTTATCATTGTG3′)。
oqrβ犪犮狋犻狀+«ueG,ö$u犜犪犃犮狋犻狀qf(5′
GACCTCACGGATAATCTAATG3′)„ 犜犪犃犮狋犻狀qr
(5′ACCATCAGGCATCTCATAG3′)。RTPCR S
}Ô\ TaKaRaÕÖjSYBRPremixEx犜犪狇TMⅡ
(TliRNaseHPlus)+,-,×ØÙ½ÚëÒۜ7
BioRadCFX96ŸÕšÉPCR觌。
1.2.4 犜犪犇27,=^_`>?Babcdefg
hijIkEF [\yz“”j犜犪犇27+«£
¤î]qrƀžŸsurveysequence(http://
www.wheatgenome.org/),“”–£¤oN.
–£¤7žŸ§jš›DO

‰ü–£¤

Ô\spidey/0|+«‚„cDNA£¤,“”+
«_DO

!£¤0|„Œ¼1j_2[MEGA6åh。Œ¼1_2Ô\Neiborjoining3


~Ô\BootstrapœŒã 。
2 õAŽ
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ÒAqr΂ƒ;RNA,S¹™ÒÔ\k^ö
$÷ƒ

“”犜犪犇27cDNA—˜,Sq4u800bp,
õ5¤SqÍ6

71)。C8—˜û–ÔyzüŸ
Œä£

‘’

n+«­®2m)•Í,j
–£¤
(GeneBank°nuKX168420„KX168421)。
\9FA™îïqr:lmžŸ¡j;<£¤


‘’

9FA™š›œ7AL„7DL§j2=>
chr7AL_4558620„chr7DL_3370387。ª¯¥¦,
7BL2=>chr7BL_6654524§©7ªlm犇27
–+«

c§?3m–+«A™T¢u犜犪犇27
7犃犔、犜犪犇277犅犔„犜犪犇277犇犔。+«_AŽ
‘’
,3m犜犪犇27+«£¤N_)•l@,¬­
®7m¯‘B,°±² •A™u828bp(7AL)、
840bp(7BL)„843bp(7DL)。HF+«_õA
ÌBCï

ÁÂÅÆB·#$)•l@

Š1)。
3ëqrD27³´£¤0|D72ë’,
HF j £ ¤ E • l @

F  a TaD277BL „
TaD277DL,=>Ì*G¶2mH+dI+jJ^。
M.NEB100bpDNAladder;1.犜犪犇27cDNA
71 犜犪犇27cDNAÜÝÞßàáâ
Fig.1 Agarosegelelectrophoresisof犜犪犇27cDNA
C1 lmnopST犇27,=q;fr
Table1 Genestructurecomparisonof犇27狊fromseveralmonocots
+«¢<
Genename
:m¯‘B •LengthofeachExon/bp
Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 Exon7
犜犪犇277犃犔 321 140 93 62 107 94 32
犜犪犇277犅犔 324 140 93 62 107 94 32
犜犪犇277犇犔 327 140 93 62 107 94 32
犅犱犇27 307 140 93 62 107 90 35
犗狊犇27 304 140 93 62 107 90 41
71317¤           4 5,Å:qrβZ[\]^_`+«š›„yzoNŠ‹AŽ
HFNKµ¬­®l˜·¸Ÿ¹º»,n(A¬L


_NAŽ‘’
,TaD27­®lm34
‰Oj DUF4033_N,n_N›œ TaD27
7AL£¤j175~256Ì*。
2.2 45犜犪犇27kstuEF
\ MEGA6/|@ž€#$jD27³´
Œ0|Ò_2Œ¼1

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73),D27³
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X+ÞxœÆbjlm
Œ¼À

qrjTaD27õYZ[ï、\]^7q
72 qrTaD27–³´j0|
Fig.2 AlignmentofwheatTaD27homologousproteins
7A_`jðaŠ’+œ500b2Øn£`jžB_cd;8efŠghir
73 qr犜犪犇27+«jü_¥jAŽ
Thefiguresatthenodesshowthebootstrapvaluesbasedon500replications,andthescalerepresentsthegeneticdistance
Fig.3 Phylogeneticanalysisofwheat犜犪犇27
8131 ! " # $ % &                   36Ó
74 qr犜犪犇27+«7€‚ƒ(A)„…†‡ˆ(B)‰jŠ‹åæ
Fig.4 Expressionpatternofwheat犜犪犇27indifferenttissues(A)andunderlowphosphatestress(B)
r

SrjklûüÏ?

TaD277ALõ\]
^7qr„SrklÏ?
,TaD277BL、TaD27
7DLõYZ[ïklÍ?,i6gqr^–mnŸ
jŒ¼op

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+«‚@–œYZ[ï
,B+«‚@–œ`qr^
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CD
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‚ƒjŠ‹ÁáoN…†‡ˆ|Š‹Éjt
u

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,犜犪犇27+«7·、·Ç„ÈÅs§
Ž‚ƒŠ‹ÉÏ)

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7
ÎjŠ‹Éυ

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Š‹ÉuЉÑ

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‡ˆÕj1/3xy,âÒ,Š‹Ézu{|×Ø,
796hÕ+,×Ø։5‡ˆÕjŠ‹É(74,
B)。õÎj}~€ja,·犜犪犇277…†‡
ˆ€‰jŠ‹ÉuЀ§0

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à

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¶ÙÚjlxy

74,B)。
3 ‚ ƒ
,.;oÁÂ犗狊犇27+«uG”,–yz{
qr犜犪犇27+«。žŸš›Š½7qr7A、
7B、7DžŸ ¡§.¶1m犜犪犇27+«„›。
+«j_AŽŠ½
,3ëqr°±²¬Y7m¯
‘B‚h

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l@

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}nõU¼+Z[\]^_j34ˆ–Íû


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ý
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BNSP1„ NSP2j¥¦Š½,ô4©7lëCx
1–犇27+«jŠ‹É@01SLsghÉjDn
—˜
[13],
iëDn—˜ô4ĊSLsj™‚ƒº
Tôåh|¯(š›}~j}œ

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¶Î £ ¤ © j I H · ï
(犜狉犻犳狅犾犻狌犿 狉犲狆犲狀狊
Linn)[14]、ÄΣ¤©—¢AM ú€—¢AM ú5օ†‡ˆÕ(A{€jSLghžŸ。Y
ó

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犇27+«jŠ‹ÉÍ|œs§(A‹…j@Ÿ
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,犜犪犇277…†‡ˆ‰jŠ‹7Î
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Ò

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,犜犪犇27jŠ‹É#¼€S,‰
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