The fluorescence observation of CaENO1-EGFP fusion protein . 29

4.2.4 The fluorescence observation of CaENO1-EGFP fusion protein

To further examine whether the EGFP fusion protein could be expressed, I incubated the transformed cells at 30 for 48 hours℃ and observed under fluorescence microscope with 400X magnification.

As shown in Figure 9, 10560-2B-f2 (shown in B) and 10560-2B-f3(shown in C) were fluorescent under fluorescence microscope. 10560-2B-f0 (shown in A) was negative control in this data. It follows that the EGFP fusion protein was fluorescent.

4.3 The analysis of truncated CaENO1

In this experiment, in order to determine which region of CaENO1 gene was critical to secretion, I designed different constructs of truncated-CaENO1 fused to EGFP and compared their activities to the control construct. The diagram of this experiment was shown in Figure 10.

The fragments of truncated CaENO1 were amplified by PCR. I designed several pairs of primers for PCR amplification of different length of truncated CaENO1. The PCR products were then treated with restriction enzymes for ligation with vector. The diagrams of constructs are shown in Figure 11.

4.3.1.1 The negative control YEP363-EGFP plasmid

For this construct, the vector and insert were ligated together to form the negative control plasmid named YEP363-EGFP in this study (shown in Figure 12A). The PCR products containing BamHI and SpeI site on 3’ and 5’end (band A in lane 1-2 of Figure 12B) as insert about 500 bp were obtained with primers ACT1-F and ACT1-R and then treated with BamHI and SpeI. The plasmid YEP363-CaENO1-EGFP, as vector, was also digested with restriction enzymes and then ligated with insert. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid YEP363-EGFP was about 9783 bp and would be digested into two DNA fragments 3643 and 6140 bp ( the band a and d in lane1-2 of Figure 13B ) by BsrGI. The plasmid was in accord with the prediction.

4.3.1.2 The YEP363- CaENO1[1-150]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/1-f and eno1/150R) and then treated by SpeI.

It is about 150 bp (the band A in lane 1 of Figure 14B). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1/[1-150]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 9945 bp and would be digested into 871 bp, 3643 bp and 5431 bp DNA fragments (b, f and g in lane 1 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.3 The YEP363- CaENO1[1-279]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/1-f and eno1/279R) by PCR and then treated by SpeI. It is about 279 bp (the band B in lane 2 of Figure 14B). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[1-279]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 10074 bp and would be digested into 1000 bp, 3643 bp and 5431 bp DNA fragments (b, f and g in lane 2 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.4 The YEP363- CaENO1[1-387]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/1-f and eno1/387R) by PCR and then treated by SpeI. It is about 279 bp (the band C in lane 3 of Figure 14B). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[1-387]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 10182 bp and would be digested into 1108 bp, 3643 bp and 5431 bp DNA fragments (d, f and g in lane 3 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.5 The YEP363- CaENO1[1-510]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/1-f and eno1/510R) by PCR and then treated by SpeI. It is about 279 bp (the band D in lane 4 of Figure 14B). The vector and insert were ligated together to form the recombinant plasmid named YEP363-

restriction enzyme digestion. The plasmid was about 10305 bp and would be digested into 397 bp, 843 bp, 3643 bp and 5431 bp DNA fragments (a, b, f and g in lane 4 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.6 The YEP363- CaENO1[280-1320]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno1/280F and eno/1320-r) by PCR and then treated by SpeI. It is about 1041 bp (the band E in lane 6 of Figure 14C). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[280-1320]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 10837 bp and would be digested into 1644 bp, 3643 bp and 5550 bp DNA fragments (h, i and j in lane 5 of Figure 15B) by BsrGI. The plasmid was in accord with the prediction.

4.3.1.7 The YEP363- CaENO1[388-1320]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno1/388F and eno/1320-r) by PCR and then treated by SpeI. It is about 933 bp (the band G in lane 7 of Figure 14C). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[388-1320]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 10729 bp and would be digested into 1644 bp, 3643 bp and 5442 bp DNA fragments (h, i and j in lane 6 of Figure 15B) by BsrGI. The plasmid was in accord with the prediction.

4.3.1.8 The YEP363- CaENO1[1-450]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end

as insert was obtained with primers (eno/1-f and eno/450-r) by PCR and then treated by SpeI. It is about 450 bp (the band H in lane 8 of Figure 14D). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[1-450]-EGFP in this study. The recombinant plasmid was checked by restriction enzyme digestion. The plasmid was about 10245 bp and would be digested into 447 bp, 774 bp, 3643 bp and 5431 bp DNA fragments (l, m, r and s in lane 7 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.9 The YEP363- CaENO1[451-900]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/451-f and eno/900-r) by PCR and then treated by SpeI. It is about 450 bp (the band H in lane 9 of Figure 14D). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[451-900]-EGFP in this study. The plasmid was about 10248 bp and would be digested into 1174 bp, 3643 bp and 5431 bp DNA fragments (n, r and s in lane 8 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.10 The YEP363- CaENO1[901-1320]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/901-f and eno/1320-r) by PCR and then treated by SpeI. It is about 420 bp (the band I in lane 10 of Figure 14D). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[901-1320]-EGFP in this study. The plasmid was about 10218 bp and would be digested into 1144 bp, 3643 bp and 5431 bp DNA fragments (n, r and s in lane 9 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/1-f and eno/900-r) by PCR and then treated by SpeI. It is about 900 bp (the band J in lane 11 of Figure 14D). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[901-1320]-EGFP in this study. The plasmid was about 10695 bp and would be digested into 397 bp, 1224 bp, 3643 bp and 5431 bp DNA fragments (k, n, r and s in lane 10 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

4.3.1.12 The YEP363- CaENO1[451-1320]-EGFP plasmid

For this construct, the YEP363-EGFP plasmid, as vector, was digested with restriction enzyme SpeI. The PCR product containing SpeI site on both 3’ and 5’end as insert was obtained with primers (eno/451-f and eno/1320-r) by PCR and then treated by SpeI. It is about 869 bp (the band K in lane 12 of Figure 14D). The vector and insert were ligated together to form the recombinant plasmid named YEP363- CaENO1[451-1320]-EGFP in this study. The plasmid was about 10668 bp and would be digested into 1594 bp, 3643 bp and 5431 bp DNA fragments (q, r, sin lane 11 of Figure 15B) by XbaΙ and BsrGI. The plasmid was in accord with the prediction.

All of above plasmids were transformed into 10560-2B strain to expressed truncated CaENO1-EGFP fusion protein. The transformed strain was named according to its target plasmid in this study; for example, the 10560-2B-CaENO1[1-150] strain was transformed by the YEP363- CaENO1[1-150]-EGFP plasmid.