Construcción de relaciones

Plants at the stage of developing 5-6 inflorescences were used for crossing. All the immature anthers around stigmata of receptor flowers where the petals just started to appear were carefully removed to avoid self-fertilization. The pollen of open flowers of donor plants was transferred to the stigmata of the emasculated plants.

4.2.5.1. Generation of homozygous lines combining the lines with different UGT76B1

expression and lines deficient in the SA pathway or the JA / ET pathway

To explore where and how UGT76B1 is integrated in the SA and JA/ET pathways, lines with different UGT76B1 expression were introgressed into lines deficient either in SA or JA/ET pathways. Homozygous lines were identified by those commercial PCR primes which can amplify the band when the T-DNA insertion is in the genome, the antibiotic markers or the Cleaved Amplified Polymorphic Sequences (CAPS) polymorphisms. The detail methods to

screen each line will be described in the following parts. The homozygous lines used in the project include ugt76b1-1 sid2, UGT76B1-OE-7 sid2, ugt76b1-1 npr1, UGT76B1-OE-7 npr1, ugt76b1-1 NahG, ugt76b1-1 jar1 and UGT76B1-OE-7 jar1.

4.2.5.2. Genotyping for double mutants

To identify the homozygous mutation of the genes containing the T-DNA insertion sequence, two primer pairs were used for the subsequent PCR in a volume of 20 µL, according to the protocol of Sigma (Extract-N-AmpTM plant PCR kit). One primer pair can recognize both the genomic DNA and the T-DNA insertion sequences and produce an amplicon with partial T- DNA sequences. The other primer pair can produce an amplicon containing only the genomic DNA sequences. The homozygous mutation was identified via observing the bands of the PCR products amplified from both primer pairs via DNA gel electrophoresis. Only the primer pair recognizing both genomic DNA and T-DNA insertion sequence can produce one band, whereas the other primer pair cannot produce any band, because of the huge T-DNA insertion. To identify mutants with point mutations causing a Cleaved Amplified Polymorphic

Sequences (CAPS) polymorphisms (www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechDCAPS.shtml), 10 µL of the PCR

reactions were digested by the restriction enzyme which recognizes CAPS polymorphisms, whereas the remaining 10 µL of the PCR reaction was used as the control. The homozygous mutation was identified by observing the pattern of the fragments depending on the particular marker analyzed (see below).

4.2.5.3. Production of the ugt76b1-1 sid2 and UGT76B1-OE-7 sid2 double mutants

The sid2-1 mutantwas introduced into ugt76b1-1 and UGT76B1-OE-7 to generate the double mutants. In sid2-1, the single nucleotide substitution of C to T in exon 9 results in the conversion Glu449STOP and diminishes the recognition site by the restriction enzyme Mfe I, thereby causing the CAPS polymorphisms. The primer pair was designed to amplify a fragment of 1112 bp in PCR with the substitution site included. In wild type, cleaving of the PCR products by Mfe I will generate two fragments of 392 bp and 720 bp, whereas the intact band of 1112 bp was observed in sid2-1. The homozygous sid2 mutation was identified according to the criterion that only one band of 1112 bp after the digestion of PCR products by Mfe I could be observed on the DNA gel. A volume of 10 µL of the 25 µL of the PCR reaction was treated with 5 units of the restriction enzyme Mfe I.

The homozygous ugt76b1 mutation was genotyped via PCR using the two pairs of primers from SALK (http://signal.salk.edu/cgi-bin/tdnaexpress) (Table in 4.1.6.1). In the F2 generation the homozygous ugt76b1 sid2 line was obtained at a ratio of 1:16. The sequence of UGT76B1 is linked together with a dominant BASTA-resistance marker in the UGT76B1 overexpression lines. To identify the UGT76B1-OE-7 sid2 double mutant, the sid2 homozygous mutation was selected among the F2 generation progeny. The plants of the F2 generation including both homozygous and heterozygous sites for the insertion of UGT76B1 were selected by the dominant BASTA resistance. The homozygous UGT76B1-OE-7 sid2 double mutant was obtained by screening non-segregating BASTA resistance in the following F3 generation.

4.2.5.4. Production of the ugt76b1-1 NahG and UGT76B1-OE-7 NahG double mutants

The transgenic NahG line expressing an SA hydroxylase and degrading total SA levels (Gaffney et al., 1993) was introgressed into ugt76b1-1 and UGT76B1-OE-7. The existence for the NahG sequence was genotyped by PCR using the primer pair designed based on NahG sequence (Table in 4.1.6.1). The homozygosity for NahG was identified without segregation in the next generation. The homozygosity for the ugt76b1 knockout and UGT76B1 overexpression were selected as above.

4.2.5.5. Production of the ugt76b1-1 npr1 and UGT76B1-OE-7 npr1 double mutants

The npr1-1 mutant causes the substitution of the single nucleotide C to T, transforming a highly conserved histidine (residue 334) to tyrosine in the third ankyrin-repeat consensus sequence. The nucleotide substitution results in the loss of the recognition site by the restriction enzyme Nla III. The primer pair was designed to amplify a 272 bp fragment including the substitution site. The PCR products from wild type were cut by Nla III, yielding a 184 bp fragment and an 83 bp fragment whereas the PCR products from the mutant npr1-1 could not be cut leading to an intact 272 bp fragment. The homozygous line of ugt76b1-1 npr1 was selected from the F2 generation. The homozygous line of UGT76B1-OE-7 npr1 was obtained in the F3 generation as above.

4.2.5.6. Production of the ugt76b1-1 jar1 and UGT76B1-OE-7 jar1 double mutant

The jar1 mutant is resistant to the inhibition of root growth by MeJA, showing longer roots on a medium containing MeJA relative to wild type (Berger, 2002). The jar1 mutant was crossed with ugt76b1-1 and UGT76B1-OE-7 to generate the ugt76b1-1 jar1 and UGT76B1- OE-7 jar1 double mutants in order to investigate the dependence of phenotypes caused by different UGT76B1 expression on JAR1. The homozygous site for the jar1 mutation was selected on ½ MS medium containing 10 µM MeJA. Then the lines with the homozygous jar1 mutation were transferred to soil and further screened for homozygosities for the ugt76b1 knockout or the UGT76B1 overexpression.