请教yong主任和各位高手,几个PCR引物设计的深层次的问题

目的：RT-PCR克隆全长目的基因做表达
基因1
1: M25157. Rat Cu, Zn supero...[gi:207011] Links

LOCUS RATSODCZL 601 bp mRNA linear ROD 27-APR-1993
DEFINITION Rat Cu, Zn superoxide dismutase mRNA, complete cds.
ACCESSION M25157
VERSION M25157.1 GI:207011
KEYWORDS superoxide dismutase.
SOURCE Rattus norvegicus (Norway rat)
ORGANISM Rattus norvegicus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae;
Rattus.
REFERENCE 1 (bases 1 to 601)
AUTHORS Hass,M.A., Iqbal,J., Clerch,L.B., Frank,L. and Massaro,D.
TITLE Rat lung Cu,Zn superoxide dismutase. Isolation and sequence of a
full-length cDNA and studies of enzyme induction
JOURNAL J. Clin. Invest. 83 (4), 1241-1246 (1989)
MEDLINE 89198078
PUBMED 2703531
COMMENT Original source text: Rat (Sprague-Dawley, adult) lung cell line JM
109, cDNA to mRNA, clone Cu,ZnSOD 13.
Draft entry and computer-readable sequence for [1] kindly provided
by M.Hass, 01-JUN-1989.
FEATURES Location/Qualifiers
source 1..601
/organism="Rattus norvegicus"
/mol_type="mRNA"
/db_xref="taxon:10116"
mRNA <1..601
/product="SOD mRNA"
CDS 59..517
/note="Cu, Zn superoxide dismutase (EC 1.15.1.1)"
/codon_start=1
/protein_id="AAA42160.1"
/db_xref="GI:207012"
/translation="MKAVCVLKGDGPVQGVIHFEQKASGEPVVVSGQITGLTEGEHGF
HVHQYGDNTQGCTTAGPHFNPHSKKHGGPADEERHVGDLGNVAAGKDGVANVSIEDRV
ISLSGEHSIIGRTMVVHEKQDDLGKGGNEESTKTGNAGSRLACGVIGIAQ"
ORIGIN 42 bp upstream of AvaI site; put. chromosome 21.
1 ggcggcttct ctcgtctcct tgctttttgc gccgcgcgtc tcccggggaa gcatggcgat
61 gaaggccgtg tgcgtgctga agggcgacgg tccggtgcag ggcgtcattc acttcgagca
121 gaaggcaagt ggtgaaccag tggtggtgtc aggacagatt acaggattaa ctgaaggcga
181 gcatgggttc catgtccatc aatatggaga caatacacaa ggctgtacca ctgcaggacc
241 tcattttaat cctcactcta agaaacatgg cggtccagcg gatgaagaga ggcatgttgg
301 agacctgggc aatgtggctg ctggaaagga cggtgtggcc aatgtgtcca ttgaagatcg
361 tgtgatctca ctctcaggag agcattccat cattggccgt actatggtgg tccacgagaa
421 acaagatgac ttgggcaaag gtggaaatga agaaagtaca aagactggaa atgctggaag
481 ccgcttggct tgtggtgtga ttgggattgc ccaataaaca ttccctatgt ggtctgagtc
541 tcagactcat ctgctggcct gctaactgta gaaaaaaacc aaaccattaa actgtaatct
601 t
为了扩增该基因做基因表达，文献报道设计的引物序列如下：
SOD(f) atg aag gcc gtg tgc gtg ct
SOD(r) att ggg caa tcc caa tca c（注：ATT可能是起终止密码子的作用，是否应为TTA）
上述两个序列经pubmed的blast比对后发现能特异的扩增大鼠SOD基因，但我发现可能存在2个问题：
（1）引物R设计时向CDS的5`末段缩进了2个碱基，因此可能会使cDNA表达时缺少终止密码子。是不是如果表达载体上的多克隆位点如果没有终止密码子或要将2个基因连接到同一载体上做2个基因的共表达时必须将终止密码子加到反向引物上？如果要加如何加？
（2）我认为克隆一个基因的全长序列做表达，不一定必须限制在CDS的两端，同时要优化ATG前的序列加上KOZAK序列才有利于后续的基因表达,因此用primer5.0设计该基因的引物序列如下：
SOD(f) 5`GGG CAG CAT GGC GAT GA 3`
SOD(r) 5`ACT CAG ACC ACA TAG GGA AT 3`
扩增区域为46-539.而CDS为59-517.这样得到的PCR产物一方面由于正向引物带有KOZAK序列,可能对基因的表达有好处,但我的ATG根据cDNA的序列好象分属不同的密码子,不知基因表达时能不能将其结合在一起,识别为ATG,而不是按照cDNA的编码序列识别为CAT GGC,从而散失作为启动子的功能.另一方面,反向引物扩增的得到PCR产物在CDS的下游,避免了设计引物时人工加上终止密码子TTA.

基因2
L25069. Mouse catalase mR...[gi:442440] Links

LOCUS MUSCATALAA 2423 bp mRNA linear ROD 10-MAY-1995
DEFINITION Mouse catalase mRNA, complete cds.
ACCESSION L25069
VERSION L25069.1 GI:442440
KEYWORDS catalase.
SOURCE Mus musculus (house mouse)
ORGANISM Mus musculus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Rodentia; Sciurognathi; Muridae; Murinae; Mus.
REFERENCE 1 (bases 1 to 2423)
AUTHORS Reimer,D.L., Bailley,J. and Singh,S.M.
TITLE Complete cDNA and 5' genomic sequences and multilevel regulation of
the mouse catalase gene
JOURNAL Genomics 21 (2), 325-336 (1994)
MEDLINE 94375055
PUBMED 8088826
COMMENT Original source text: Mus musculus (strain BALB/c, sub_species
domesticus) (library: Stratagene) adult liver cDNA to mRNA.
FEATURES Location/Qualifiers
source 1..2423
/organism="Mus musculus"
/mol_type="mRNA"
/strain="BALB/c"
/sub_species="domesticus"
/db_xref="taxon:10090"
/tissue_type="liver"
/dev_stage="adult"
/tissue_lib="Stratagene"
5'UTR 1..87
CDS 88..1671
/codon_start=1
/product="catalase"
/protein_id="AAA66054.1"
/db_xref="GI:442441"
/translation="MSDSRDPASDQMKQWKEQRASQRPDVLTTGGGNPIGDKLNIMTA
GSRGPLLVQDVVFTDEMAHFDRERIPERVVHAKGAGAFGYFEVTHDITRYSKGKVFEH
IGKRTPIAVRFSTVAGESGSADTVRDPRGFAVKFYTEDGNWDLVGNNTPIFFIRDAIL
FPSFIHSQKRNPQTHLKDPDMVWDFWSLRPESLHQVSFLFSDRGIPDGHRHMNGYGSH
TFKLVNADGEAVYCKFHYKTDQGIKNLPVGEAGRLAQEDPDYGLRDLFNAIANGNYPS
WTFYIQVMTFKEAETFPFNPFDLTKVWPHKDYPLIPVGKVVLNKNPVNYFAEVEQMAF
DPSNMPPGIEPSPDKKLQGRLFAYPDTHRHRLGPNYLQIPVNCPYRARVANYQRDGPM
CMHDNQGGAPNYYPNSFSAPEQQRSALEHSVQCAVDVKRFNSANEDNVTQVRTFYTKV
LNEEERKRLCENIAGHLKDAQLFIQKKAVKNFTDVHPDYGARIQALLDKYNAEKPKNA
IHTYTQAGSHMAAKGKANL"
3'UTR 1672..2423
repeat_unit 2062..2142
/function="'mRNA protein binding'"
/evidence=experimental
/label=CA31
repeat_unit 2174..2189
/function="'mRNA protein binding'"
/evidence=experimental
/label=T15
repeat_unit 2273..2307
/label=TGTGC7
polyA_signal 2404..2410
ORIGIN
1 attgccttct ccgggtggag accagaccgc tgcgtccgtc cctgctgtct cacgttccgc
61 agctctgcag ctccgcaatc ctacaccatg tcggacagtc gggacccagc cagcgaccag
121 atgaagcagt ggaaggagca gcgggcctcg cagagacctg atgtcctgac caccggaggc
181 gggaacccaa taggagataa acttaatatc atgaccgcgg ggtcccgagg gcccctcctc
241 gttcaggatg tggttttcac tgacgagatg gcacactttg acagagagcg gattcctgag
301 agagtggtac acgcaaaagg agcaggtgct tttggatact ttgaggtcac ccacgatatc
361 accagatact ccaagggaaa ggtgtttgag catattggaa agaggacccc tattgccgtt
421 cggttctcca cagtcgctgg agagtcaggc tcagctgaca cagttcgtga ccctcggggg
481 tttgcagtga aattttacac tgaagatggt aactgggatc ttgtgggaaa caacacccct
541 attttcttca tcagggatgc catattgttt ccatccttta tccatagcca gaagagaaac
601 ccacagactc acctgaagga tcctgacatg gtctgggact tctggagtct tcgtcccgag
661 tctctccatc aggtttcttt cttgttcagt gaccgaggga ttcccgatgg tcaccggcac
721 atgaatggct atggatcaca caccttcaag ttggttaatg cagatggaga ggcagtctat
781 tgcaagttcc attacaagac cgaccagggc atcaaaaact tgcctgttgg agaggcagga
841 aggcttgctc aggaagatcc ggattatggc ctccgagatc ttttcaatgc catcgccaat
901 ggcaattacc cgtcctggac gttttacatc caggtcatga cttttaagga ggcagaaact
961 ttcccattta atccatttga tctgaccaag gtttggcctc acaaggacta ccctcttata
1021 ccagttggca aagtggtttt aaacaaaaat ccagttaatt actttgctga agttgaacag
1081 atggcttttg acccaagcaa tatgccccct ggcatcgagc ccagccctga caaaaagctt
1141 cagggccgcc tttttgccta cccggacact caccgccacc gcctgggacc caactatctg
1201 cagatacctg tgaactgtcc ctaccgcgct cgagtggcca actaccagcg tgatggcccc
1261 atgtgcatgc atgacaacca gggtggtgcc cccaactatt accccaacag cttcagcgca
1321 ccagagcagc agcgctcagc cctggagcac agcgtccagt gcgctgtaga tgtgaaacgc
1381 ttcaacagtg ctaatgaaga caatgtcact caggtgcgga cattctacac aaaggtgttg
1441 aatgaggagg agaggaaacg cctgtgtgag aacattgccg gccacctgaa ggacgctcag
1501 cttttcattc agaagaaagc ggtcaagaat ttcactgacg tccaccctga ctatggggcc
1561 cgcatccagg ctcttctgga caagtacaac gctgagaagc ctaagaacgc aattcacacc
1621 tacacgcagg ccggctctca catggctgcg aagggaaaag ctaacctgta actccggtgc
1681 tcagcctccg ctgaggagac ctctcgtgaa gccgagcctg aggatcacct gtaatcaacg
1741 ctggatggat tctcccccgc cggagcgcag actcacgctg atgactttaa aacgataatc
1801 cgggcttcta gagtgaatga taaccatgct tttgatgccg tttcctgaag ggaaatgaaa
1861 ggttagggct tagcaatcat ttaacagaaa catggatcta ataggacttc tgtttggatt
1921 attcatttaa atgactacat ttaaaatgat tacaagaaag gtgttctagc cagaaacatg
1981 acttgattag acaagataaa aatcttggcg agaatagtgt attctcctat tacctcatgg
2041 tctggtatat atacaataca acacacatac cacacacaca cacacatgca atacacacac
2101 tacacacaca tacacacact cacacacact catacacaca catgaagaga tgataaagat
2161 ggcccactca gaattttttt tttatttttc taaggtcctt ataagcaaaa ccatacttgc
2221 atcatgtctt ccaaaagtaa ctttagcact gttgaaactt aatgtttatt cctgtgctgt
2281 gcggtgctgt gctgtgctgt gctgtgcagc taatcagatt cttgtttttt cccacttgga
2341 ttatgttgat gctaatacgc agtgatttca cataggatga tttgtacttg cttacatttt
2401 tacaataaaa tgatctacat gga
通过PRIMRE5.0软件,将该基因的CDS上游约200BP和下游约200BP的全部序列进行搜索,没有合适的引物能直接从ATG-TAA直接扩增该基因的全长,因此只好设计如下的引物进行部分扩增:
正向引物序列:5` AGC GAC CAG ATG AAG CAG 3`
反向引物序列:5`AGC CAT GTG AGA GCC GG 3`
扩增的范围在CDS的ATG 下游16BP,其RT-PCR产物编码氨基酸序列的C端缺少5个氨基酸,N端缺少7个氨基酸.不知这样设计引物可行吗?缺少的氨基酸对该蛋白质的影响大不大?但这样设计是可在正向引物序列中有效的存在KOZAK序列,可能有利于基因表达。我不能确定的是反向引物序列是否要在5`端加入终止密码子TAA、TAG或TGA。