Table 3.
Evaluation of 12 putative transgenic maize lines for transformation and PR10 expression at transcript and protein levels.
| Line name | Transformation* | PR10 expression in leaf†,‡ | PR10 expression in root†,‡ | PR10 level in mature kernels§ | ||
|---|---|---|---|---|---|---|
| ΔCt | Changes (%) | ΔCt | Changes (%) | |||
| A44S1‐1‐1 | Y | 19.250 cd | −47.8 | 15.905 b | −87.4 | −11.2% |
| A44S1‐1‐4 | Y | 17.480 e | 78.2 | 12.752 efg | 11.9 | −30.3% |
| A44S1‐3‐1 | Y | 20.735 ab | −81.3 | 13.705 de | −42.2 | −78.8%¶ |
| A44S1‐3‐4 | Y | 21.380 a | −88.1 | 15.697 b | −85.5 | −65.4%¶ |
| A44S1‐5‐2 | Y | 19.813 bc | −64.6 | 13.475 def | −32.1 | −85.1%¶ |
| A44S1‐5‐3 | Y | 19.703 bc | −61.8 | 14.407 cd | −64.4 | −61.3%¶ |
| A44S1‐6‐1 | Y | 18.320 de | −0.5 | 13.585 def | −37.2 | −39.2% |
| A44S1‐15‐3 | N | 17.053 e | 139.5 | 11.185 h | 231.7 | −6.5% |
| A44S1‐35‐3 | Y | 19.038 cd | −39.5 | 17.480 a | −95.8 | −75.7%¶ |
| A44S1‐35‐4 | Y | 21.173 a | −86.2 | 14.190 cd | −58.7 | −63.2%¶ |
| A44S1‐36‐1 | Y | 19.228 cd | −49.1 | 13.670 de | −40.8 | −64.5%¶ |
| A44S1‐42‐1 | Y | 19.255 cd | −47.9 | 15.287 bc | −80.7 | −84.2%¶ |
| HT40917‐1 | N | 18.425 de | −7.4 | 12.912 efg | 0.0 | 0.0 |
| HT40917‐3 | N | 18.210 de | 7.4 | 12.922 efg | −0.5 | ND |
Transformation was verified through polymerase chain reaction (PCR) with PR10if (GTTCAACTTCACCTCAG G) and PR10ir (AAGCTGAACGGCATGACT) primers corresponding to the chloramphenical resistance gene (CmR) region spanned by the intron using the genomic DNA isolated from seedling leaves of the corresponding transgenic lines. Kernels from non‐transgenic Hi II plants were used as a negative control (HT40917‐1). ‘Y’ and ‘N’ indicate positive and negative for transgene, respectively.
The PR10 transcript level was determined using real‐time PCR with 18S as an internal normalizer. The percentage change in target gene is calculated as the difference in relative expression between RNAi‐silenced transgenic and control, expressed as a percentage of the control. The relative expression level is calculated on the basis of the threshold cycle (Ct) values of the target gene and the 18S normalizer as follows: relative expression level = power (2, ΔCt), where ΔCt=Ct (18S rRNA)−Ct (target). The data presented here are means combined from two experiments.
Means within the column followed by the same letter were not significantly different based on Waller–Dunncan K‐ratio t‐test (at P= 0.05).
PR10 protein reduction was determined using Progenesis gel analysis software based on four gels (two repeated experiments with two replicated gels each).
Indicates the reduction was significant compared with the control (HT40917‐1) based on least‐significant difference (P= 0.05). ND, not determined.