RT-PCR Amplification of Putative ACC Synthase Gene Transcripts

Previously, RT-PCR had been used to isolate and clone three TR-ACS genes from the white clover genotype 10F of cultivar Grasslands Challenge (Murray, 2001). Similar procedures were then used in this thesis to isolate orthologues and novel TR-ACS

genes from the white clover ecotype Tienshan and cultivar Kopu.

Initially, ACC synthase cDNAs were amplified by RT-PCR using nested degenerate oligonucleotide primers (Section 2.5.5.1.) corresponding to conserved sequences within the ACS genes (boxes I to VI, Figure 4.3.). The first round primers (ACSR1F and ACSR6R) generated cDNA transcripts of approximately 780 bp from the total cDNA pool, which represented ACS transcripts within the conserved boxes I to VI. An aliquot of the first round PCR products were then used as templates for the second round of PCR amplification using ACSR2F and ACSR6R primers. This second round PCR amplified products that corresponded to sequences between conserved boxes II to VI.

0 2 4 6 8 10 12 5 10 15 20 25 30

Soil w ater content (%)

P e tio le e lo n g a tio n r a te ( m m /d a y ) 1 2 3 4 5

Results 2

122 Figure 4.3 Diagrammatic representation of an ACC synthase gene from

Arabidopsis thaliana (modified from Murray, 2001).

The boxed regions denote the exons and lines denote the introns; conserved regions are denoted by the dark boxes; RC, the reactive centre; A, polyadenylation signal. The position of degenerate primers for the amplification of ACC synthase genes in white clover are shown as: first round forward primer (F1), second round forward primer (F2) and reverse primer (R6).

Figure 4.4 RT-PCR using RNA isolated from apical structures and first-fully expanded leaves of the Tienshan ecotype harvested at different soil water contents.

Nested degenerate primers were used for two rounds of PCR amplification. The approximate size of the amplified cDNA is indicated.

Lane 1. DNA Ladder

Lanes 2 to 6. RT-PCR products amplified from RNA isolated from apical structures at ca. 28%, 20%, 18%, 8% and 6% SWC respectively.

Lanes 7 to 11. RT-PCR products amplified from RNA isolated from first-fully expanded leaves at ca.28%, 20%, 18 %, 8% and 6% SWC.

780 bp 1800 bp F1 F2 R6 1 2 3 4 5 6 7 RC _A -670 bp Lane 1 2 3 4 5 6 7 8 9 10 11 670 bp

Results 2

123 The amplified first round PCR products (ca. 780 bp) were unable to be detected after electrophoresis on a 1% (w/v) agarose gel and ethidium bromide staining (data not shown). However, amplified products from the second round PCR (ca. 670 bp) were detected and examples are shown as Figure 4.4.

The second round PCR products (Figure 4.4.) were TA-cloned into the pGEM T- easy vector and transformed into the E. coli strain DH5, and putative inserts detected by blue/white screening (Figure 4.5).

Figure 4.5 White-blue colony selection of sub-libraries generated from the apical structures of the Tienshan ecotype harvested at 8% SWC. Colonies were grown in LB media supplemented with IPTG and X-Gal.

Initially, only white colonies were selected and cultured in LB Amp100 _broth,

plasmids isolated (Section 2.5.7.2) and the presence of inserts confirmed by PCR, using M13 primers, and the DNA sequences then obtained. All of the DNA sequences obtained from this protocol belonged to two genes, which have high sequence identity to the TR-ACS1 and TR-ACS2 sequences of white clover genotype 10F, Cultivar Grasslands Challenge (Murray, 2001). These results suggested that TR- ACS3 and any novel TR-ACS genes may not occur frequently (or at all) in apical tissues, or they might have been present in the blue or pale blue colonies. Since blue colonies could also contain the insert, as a result of in-frame cloning into the LacZ

Results 2

124 gene or the introduction of a mutation during the amplification process, no further blue/white colony selection was carried out.

For subsequent clones, the presence of inserts of approximately 670 bp was determined by PCR from all colonies without selection. To perform this, PCR was carried out directly from the colonies (without prior plasmid isolation) using the second round ACS degenerate primer sets (ACSR2F and ACSR6R), prior to separation by 1% (w/v) agarose gel electrophoresis and visualization following ethidium bromide staining (Figure 4.6.A). For further identification and screening, the clones were rescued by sub-culturing onto a new LB Amp100 _{plate and numbered.}

Clones containing inserts were further screened for the presence of TR-ACS1 or TR- ACS2, using gene-specific primers (Section 2.5.5.1.).

Positive clones which were not amplified by the gene-specific TR- ACS1 or TR-ACS2

primers could therefore contain either the TR-ACS3 gene or a novel TR-ACS gene not previously identified by Murray (2001). Selections of these are shown in Figure 4.6, and the frequency of the genes isolated is summarised in Tables 4.1 to 4.4.

TR-ACS1 was the predominant clone found in the apical structures and first fully expanded leaves of both the Tienshan ecotype (546 and 641 clones in the apical structures and first-fully expanded leaves, respectively) and the Kopu cultivar (430 and 351 clones in the apical structures and first-fully expanded leaves, respectively). Next was TR-ACS2 (67 and 133 clones in the apical structures and first-fully expanded leaves of Tienshan, respectively), and 56 and 88 clones in the apical structures and first-fully expanded leaves of Kopu cultivar, respectively. Further, TR- ACS1 and TR-ACS2 were found in clones amplified from RNA isolated from both varieties in all SWC conditions (fully-hydrated and water-deficit conditions) (Table 4.1., 4.2., 4.3. and 4.4.).

Results 2

125 Figure 4.6 PCR selection of putative ACC synthase gene fragments.

A. Amplification of ACC synthase fragments using ACSR2F and ACSR6R as primers from sub-libraries generated from RNA isolated from apical structures of Tienshan ecotype at ca. 8% SWC. B. Amplification of TR-ACS1 from clones positively amplified in plate A using ACS1F and ACSR6R primers. C. Amplification of TR-ACS2 from clones in plate A using ACS2F and ACSR6R as primers. PCR products were separated on a 1% (w/v) agarose gel and visulalised with ethidium bromide. The molecular weights of standards are indicated on the left. The size of PCR products is indicated on the right.

Lane 1. DNA Ladder

Lanes 2 to 14 are amplified products from colonies number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 respectively. ~670 bp Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C ~670 bp ~670 bp

Results 2

126 Table 4.1 Summary of the ACC synthase clones identified by PCR and sequence analysis in the apical structures from plants of the Tienshan ecotype harvested at different soil water contents (SWCs)

% SWC TR-ACS1 TR-ACS2 TR-ACS3 TR-ACS4 Total clones

28 82 7 - - 89 20 79 4 - - 83 18 114 9 - 123 8 131 27 1 2 161 6 140 20 - - 160 Total clones 546 67 1 2 616

Table 4.2 Summary of the ACC synthase clones identified by PCR and sequence analysis in the first-fully-expanded leaves from plants of the Tienshan ecotype harvested at different soil water contents (SWCs)

% SWC TR-ACS1 TR-ACS2 TR-ACS3 TR-ACS4 Total clones

28 48 22 - - 70 20 113 31 1 - 145 18 142 22 6 - 170 8 178 19 5 - 201 6 160 39 10 - 209 Total clones 641 133 22 - 796

Table 4.3 Summary of the ACC synthase clones identified by PCR and sequence analysis in the apical structures from plants of cv. Kopu harvested at different soil water contents (SWCs)

% SWC TR-ACS1 TR-ACS2 TR-ACS3 TR-ACS4 Total clones

28 24 10 - - 34 22 79 4 - - 83 18 169 12 - 181 11 98 22 - - 120 8 60 8 - - 68 Total clones 430 56 - - 486

Results 2

127 Table 4.4 Summary of the ACC synthase clones identified by PCR and sequence