几种拟南芥突变体鉴定方法

HY5‐215

The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus‐induced development of root and hypocotyl, Genes Dev. 1997 Nov 15; 11(22): 2983–2995.

In the genome of hy5‐215, the splicing acceptor site of the first intron (=G) was replaced by A, suggesting that this mutation causes aberrant RNA processing

In hy5‐215, the nucleotide g‐1117 (white letter), which is the last nucleotide in the first intron, is replaced by an a

HY5‐215的突变位点与野生型相比，并没有酶切位点的变化。引物在有一个错配位点的情况下，可以产生一个新的酶切位点PsiI，从而将野生型、杂合、纯合进行区分。

Design proper primers and choose proper a enzyme by dCAPS Finder 2.0

(/dcaps/dcaps.html)

HY5proF GAGAGAATATGCGAGTGAATGAC Len 22 TM 54 HY5proR TCTAAAGTCTCTTTTATGTTTTA T A Len 25 TM 50.8

PsiI:

但是，实验室并没有PsiI ，所以只能再去寻找新的内切酶。在设计的引物有2

个错配位点时，可以产生新的酶切位点，AluI 将野生型切断。

HY5-215 F CGTATCTCCTCATCGCTTTCAATAG Len 25 TM 60.0 HY5-215 R GTCCCGCTCTTTTCCTCTTTATC Len 23 TM 60.8

AluI:

MYCT

The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses, Plant Cell. 2011 Feb; 23(2): 701–715.

MYC2

Mutagen : T‐DNA insertion

Insertion Flanking

Sequence:

TAAAACCGCCGGAGAATCAGATCACTCCGATCTAGAAGCT

(Length:40)

根据T‐DNA PrimerDesign（/tdnaprimers.2.html）设计引物

PRODUCT_SIZE 1186

Myc2LP TGGTTTTTCTTGGTTTCGATG Len 21 TM 59.96

Myc2RP CTCTAATCATTGCGTCCCAAC Len 21 TM 59.58

LBb1.3 ATTTTGCCGATTTCGGAAC BP+RP_PRODUCT_SIZE 558‐858

MYC3

Mutagen : T‐DNA insertion

myc3 F AAGGTGGGTTGTTGAAATCTAATG Len 24 TM 58.3

myc3 R GTTTTCTCCGACTTTCGTCATCA Len 23 TM 61

T-DNA ATATTGACCATCATACTCATTGC Len 23 TM 55.2

MYC4

Mutagen : T‐DNA insertion

myc4 F TCTCTCACAACTTGATCCAGCTAA Len 24 TM 60.0

myc4 R TAACCGATTACCATCTCAACCAA Len 23 TM 59.2

T-DNA ATATTGACCATCATACTCATTGC Len 23 TM 55.2

Phyb‐9

Mutations in the gene for the red/far‐red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell v.5(2); 1993 Feb

hy3-EMS742 is a G-toA mutation

phyb9 F CTGTTCAATCGCAGAAACTCGCGGT Len25

phyb9 R CCGTCACATTTCACTAAGTCCAT Len 23 TM58.6

MnlI:

但是，实验室并没有MnlI，所以只能再去寻找新的内切酶。在设计的引物有1个错配位点时，可以产生新的酶切位点，AluI将突变型切断。

phyb-9 F TCCAGCGAGGTGGTTACATTCAG Len 23 TM 63.9

phyb-9 R AAGTGTGATGGCAAACAACCAAAGC Len 25 TM 64

AluI:

COP1‐4

The weak copl-4 allele represents a C-to T mutation that changes the

Gln-283 CAA codon to a UAA stop codon.

cop1‐4的突变位点与野生型相比，并没有酶切位点的变化。引物在有一个错配位点的情

况下，可以产生一个新的酶切位点MaeIII，可以将突变体的切断，从而将野生型、杂合、纯合进行区分。

cop1--4 F GGAAGCACTACAAAGGGTCGGTT TM 63.7

cop1--4 R TGAGACAGTTGACTGATTCAAACGTT TM 61.9

MaeIII:

但是，实验室并没有MaeIII，所以只能再去寻找新的内切酶。在设计的引物有2个错配

位点时，可以产生新的酶切位点，MluI将突变型切断。

cop1‐4 F GGATGCGCTGAGTGGGTCAGACGCG Len 25 TM 72.0

cop1‐4 R TCACCTTGATACAATGTTGGCTG Len 23 TM 60.3

MluI: